Influence of breast cancer risk factors on proliferation and DNA damage in human breast glandular tissues: role of intracellular estrogen levels, oxidative stress and estrogen biotransformation.

Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC- and UHPLC-MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2).

Influence of breast cancer risk factors and intramammary biotransformation on estrogen homeostasis in the human breast.

Understanding intramammary estrogen homeostasis constitutes the basis of understanding the role of lifestyle factors in breast cancer etiology. Thus, the aim of the present study was to identify variables influencing levels of the estrogens present in normal breast glandular and adipose tissues (GLT and ADT, i.e., 17ß-estradiol, estrone, estrone-3-sulfate, and 2-methoxy-estrone) by multiple linear regression models. Explanatory variables (exVARs) considered were (a) levels of metabolic precursors as well as levels of transcripts encoding proteins involved in estrogen (biotrans)formation, (b) data on breast cancer risk factors (i.e., body mass index, BMI, intake of estrogen-active drugs, and smoking) collected by questionnaire, and (c) tissue characteristics (i.e., mass percentage of oil, oil%, and lobule type of the GLT). Levels of estrogens in GLT and ADT were influenced by both extramammary production (menopausal status, intake of estrogen-active drugs, and BMI) thus showing that variables known to affect levels of circulating estrogens influence estrogen levels in breast tissues as well for the first time. Moreover, intratissue (biotrans)formation (by aromatase, hydroxysteroid-17beta-dehydrogenase 2, and beta-glucuronidase) influenced intratissue estrogen levels, as well. Distinct differences were observed between the exVARs exhibiting significant influence on (a) levels of specific estrogens and (b) the same dependent variables in GLT and ADT. Since oil% and lobule type of GLT influenced levels of some estrogens, these variables may be included in tissue characterization to prevent sample bias. In conclusion, evidence for the intracrine activity of the human breast supports biotransformation-based strategies for breast cancer prevention. The susceptibility of estrogen homeostasis to systemic and tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the environment possible.

Qualitative and quantitative differences in estrogen biotransformation in human breast glandular and adipose tissues: implications for studies using mammary biospecimens.

Because of its assumed role in breast cancer etiology, estrogen biotransformation (and interaction of compounds therewith) has been investigated in human biospecimens for decades. However, little attention has been paid to the well-known fact that large inter-individual variations exist in the proportion of breast glandular (GLT) and adipose (ADT) tissues and less to adequate tissue characterization. To assess the relevance of this, the present study compares estrogen biotransformation in GLT and ADT. GLT and ADT were isolated from 47 reduction mammoplasty specimens derived from women without breast cancer and were characterized histologically and by their percentages of oil. Levels of 12 unconjugated and five conjugated estrogens were analyzed by GC- and UHPLC-MS/MS, respectively, and levels of 27 transcripts encoding proteins involved in estrogen biotransformation by Taqman® probe-based PCR. Unexpectedly, one-third of specimens provided neat GLT only after cryosection. Whereas 17ß-estradiol, estrone, and estrone-3-sulfate were detected in both tissues, estrone-3-glucuronide and 2-methoxy-estrone were detected predominately in GLT and ADT, respectively. Estrogen levels as well as ratios 17ß-estradiol/estrone and estrone-3-sulfate/estrone differed significantly between GLT and ADT, yet less than between individuals. Furthermore, estrogen levels in GLT and ADT correlated significantly with each other. In contrast, levels of most transcripts encoding enzymes involved in biotransformation differed more than between individuals and did not correlate between ADT and GLT. Thus, mixed breast tissues (and plasma) will not provide meaningful information on local estrogen biotransformation (and interaction of compounds therewith) whereas relative changes in 17ß-estradiol levels may be investigated in the more abundant ADT.

Novel insight in estrogen homeostasis and bioactivity in the ACI rat model of estrogen-induced mammary gland carcinogenesis.

Despite being widely used to investigate 17ß-estradiol (E2)-induced mammary gland (MG) carcinogenesis and prevention thereof, estrogen homeostasis and its significance in the female August Copenhagen Irish (ACI) rat model is unknown. Thus, levels of 12 estrogens including metabolites and conjugates were determined mass spectrometrically in 38 plasmas and 52 tissues exhibiting phenotypes ranging from normal to palpable tumor derived from a representative ACI study using two different diets. In tissues, 40 transcripts encoding proteins involved in estrogen (biotrans)formation, ESR1-mediated signaling, proliferation and oxidative stress were analyzed (TaqMan PCR). Influence of histo(patho)logic phenotypes and diet on estrogen and transcript levels was analyzed by 2-way ANOVA and explanatory variables influencing levels and bioactivity of estrogens in tissues were identified by multiple linear regression models. Estrogen profiles in tissue and plasma and the influence of Hsd17b1 levels on intra-tissue levels of E2 and E1 conclusively indicated intra-mammary formation of E2 in ACI tumors by HSD17B1-mediated conversion of E1. Proliferation in ACI tumors was influenced by Egfr, Igf1r, Hgf and Met levels. 2-MeO-E1, the only oxidative estrogen metabolite detected above 28-42 fmol/g, was predominately observed in hyperplastic tissues and intra-tissue conversion of E1 seemed to contribute to its levels. The association of the occurrence of 2-MeO-E1 with higher levels of oxidative stress observed in hyperplastic and tumor tissues remained equivocal. Thus, the present study provides mechanistic explanation for previous and future results observed in the ACI model.

Quinolone Amides as Antitrypanosomal Lead Compounds with In Vivo Activity.

Human African trypanosomiasis (HAT) is a major tropical disease for which few drugs for treatment are available, driving the need for novel active compounds. Recently, morpholino-substituted benzyl amides of the fluoroquinolone-type antibiotics were identified to be compounds highly active against Trypanosoma brucei brucei Since the lead compound GHQ168 was challenged by poor water solubility in previous trials, the aim of this study was to introduce structural variations to GHQ168 as well as to formulate GHQ168 with the ultimate goal to increase its aqueous solubility while maintaining its in vitro antitrypanosomal activity. The pharmacokinetic parameters of spray-dried GHQ168 and the newly synthesized compounds GHQ242 and GHQ243 in mice were characterized by elimination half-lives ranging from 1.5 to 3.5 h after intraperitoneal administration (4 mice/compound), moderate to strong human serum albumin binding for GHQ168 (80%) and GHQ243 (45%), and very high human serum albumin binding (>99%) for GHQ242. For the lead compound, GHQ168, the apparent clearance was 112 ml/h and the apparent volume of distribution was 14 liters/kg of body weight (BW). Mice infected with T. b. rhodesiense (STIB900) were treated in a stringent study scheme (2 daily applications between days 3 and 6 postinfection). Exposure to spray-dried GHQ168 in contrast to the control treatment resulted in mean survival durations of 17 versus 9 days, respectively, a difference that was statistically significant. Results that were statistically insignificantly different were obtained between the control and the GHQ242 and GHQ243 treatments. Therefore, GHQ168 was further profiled in an early-treatment scheme (2 daily applications at days 1 to 4 postinfection), and the results were compared with those obtained with a control treatment. The result was statistically significant mean survival times exceeding 32 days (end of the observation period) versus 7 days for the GHQ168 and control treatments, respectively. Spray-dried GHQ168 demonstrated exciting antitrypanosomal efficacy.

The mycotoxin patulin reacts with DNA bases with and without previous conjugation to GSH: implication for related α,ß-unsaturated carbonyl compounds?

The α,ß-unsaturated carbonyl group is recognized as alert for mutagenicity, attributed to (1) its direct reaction with DNA, counteractable by glutathione (GSH), and (2) oxidative stress caused indirectly by GSH depletion. Accordingly, the α,ß,γ,δ-unsaturated lactone patulin (PAT), a mycotoxin detected in fruits and products derived thereof, is known to induce gene, chromosome, and genome mutations in vitro, its mutagenicity correlating inversely with intracellular GSH levels. Thus, the reactivity of PAT against DNA bases and nucleosides in the absence and presence of GSH and glutathione S-transferases (GSTs) was investigated under cell-free conditions using HPLC mass spectrometry techniques for identification of reaction products. Adduct formation with all four nucleobases as well as with purine base nucleosides occurred even in the presence of GSH, revealing several adducts of PAT, mono- and disubstituted with nucleobases/nucleosides as well as novel GSH-PAT adducts. In addition, novel mixed GSH-PAT-nucleobase adducts were observed. These adducts exhibited a ketohexanoic acid-type structure of the PAT molecule, C6 substituted with GSH and linking C1 of PAT with nitrogens of nucleobases/nucleosides via an amide bond. Formation of GSH-PAT-adenine adducts was not prevented by GSTs, and excess of GSH needed to reduce their formation was higher than for PAT-adenine adducts. The formation of mixed GSH-DNA base adducts has not been described for PAT or any other α,ß-unsaturated carbonyl before, although the reaction mechanism seems to be applicable to a variety of α,ß-unsaturated carbonyls occurring in food and in the environment.

The isoflavone irilone contributes to the estrogenic potential of dietary supplements containing red clover.

A recent intervention study demonstrated the occurrence of irilone as second most abundant isoflavone next to daidzein in human plasma after consumption of a red clover-based dietary supplement (RCDS) containing predominately formononetin ≫ biochanin A > irilone (12 % of these isoflavones). To elucidate the relevance of this finding, in the present study (1) the representativeness of the isoflavone composition of the RCDS and (2) the estrogenic activity of irilone were investigated. Thus, major isoflavones were quantified in eight commercially available RCDS. Furthermore, the estrogenic activities of irilone and other isoflavones were determined by marker gene expression in Ishikawa and cell proliferation in MCF-7 cells. Irilone amounted to 1.8-10.9 mg/g capsule content and 5-18 % of the three major isoflavones, respectively, demonstrating the general occurrence of irilone in RCDS. Moreover, irilone significantly induced the activity of alkaline phosphatase (AlP) as well as AlP, progesterone receptor, and androgen receptor mRNA levels in Ishikawa cells. Furthermore, irilone significantly induced MCF-7 cell proliferation. Neither 17ß-estradiol (E2)-induced AlP activity nor E2-induced MCF-7 cell proliferation was affected by irilone. ICI182,780 antagonized IRI-induced effects on both AlP activity and cell proliferation, suggesting an estrogen receptor agonistic mode of action. Taking into account the estrogenic activity of red clover isoflavones (formononetin, biochanin A, prunetin, glycitein) and their biotransformation products (daidzein, genistein, ethylphenol) as well as published plasma levels of isoflavones after consumption of RCDS, irilone could contribute approximately 50 % of the E2 equivalents estimated for daidzein.

A primaquine-chloroquine hybrid with dual activity against Plasmodium liver and blood stages.

We present a new class of hybrid molecules consisting of the established antiplasmodial drugs primaquine and chloroquine. No drug is known to date that acts comparably against all stages of Plasmodium in its life cycle. Starting from available precursors, we designed and synthesized a new-generation compound consisting of both primaquine and chloroquine components, with the intent to produce agents that exhibit bioactivity against different stages of the parasite's life cycle. In vitro, the hybrid molecule 3 displays activity against both asexual and sexual P. falciparum blood stages as well as P. berghei sporozoites and liver stages. In vivo, the hybrid elicits activity against P. berghei liver and blood stages. Our results successfully validate the concept of utilizing one compound to combine different modes of action that attack different Plasmodium stages in the mammalian host. It is our hope that the novel design of such compounds will outwit the pathogen in the spread of drug resistance. Based on the optimized synthetic pathway, the compound is accessible in a smooth and versatile way and open for potential further molecular modification.

Validation of a GC- and LC-MS/MS based method for the quantification of 22 estrogens and its application to human plasma.

In epidemiological studies, blood levels of 17ß-estradiol (E2) are associated with hormone-dependent diseases. The lack of specific methods impedes studies on the role of E2 metabolites and their conjugates in the etiology of hormone-dependent diseases. Stable-isotope dilution tandem mass spectrometry methods (coupled to gas chromatography and liquid chromatography systems) for the analysis of 22 endogenous estrogens, including both oxidative metabolites, as well as sulfates and glucuronides, was validated and the method applied to plasma of women with no breast cancer. No changes in estrogen profile during sample cleanup were observed and values for limit of detection (7fmol/ml - 2 pmol/ml), accuracies (80-122%) as well as intra- and inter-day precision (below 28%) at levels near the limit of quantification were acceptable. In human plasma only seven estrogens were detected and estrone conjugates contributed most to the estrogen profile.

Evaluation of the test accuracy of a SARS-CoV-2 rapid antigen test in symptomatic community dwelling individuals in the Netherlands.

BACKGROUND: SARS-CoV-2 real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is well suited for the diagnosis of clinically ill patients requiring treatment. Application for community testing of symptomatic individuals for disease control purposes however raises challenges. SARS-CoV-2 rapid antigen tests might offer an alternative, but quality evidence on their performance is limited. METHODS: We conducted an evaluation of the test accuracy of the 'BD Veritor System for Rapid Detection of SARS-CoV-2' (VRD) compared to qRT-PCR on combined nose/throat swabs obtained from symptomatic individuals at Municipal Health Service (MHS) COVID-19 test centers in the Netherlands. In part one of the study, with the primary objective to evaluate test sensitivity and specificity, all adults presenting at one MHS test center were eligible for inclusion. In part two, with the objective to evaluate test sensitivity stratified by Ct (cycle threshold)-value and time since symptom onset, adults who had a positive qRT-PCR obtained at a MHS test center were eligible. FINDINGS: In part one (n = 352) SARS-CoV-2 prevalence was 4.8%, overall specificity 100% (95%CI: 98·9%-100%) and sensitivity 94·1% (95%CI: 71·1%-100%). In part two (n = 123) the sensitivity was 78·9% (95%CI: 70·6%-85·7%) overall, 89·4% (95% CI: 79·4%-95·6%) for specimen obtained within seven days after symptom onset and 93% (95% CI: 86%-97.1%) for specimen with a Ct-value below 30. INTERPRETATION: The VRD is a promising diagnostic for COVID-19 testing of symptomatic community-dwelling individuals within seven days after symptom onset in context of disease control. Further research on practical applicability and the optimal position within the testing landscape is needed.

The scent of the waggle dance.

The waggle dance of honey bee (Apis mellifera L.) foragers communicates to nest mates the location of a profitable food source. We used solid-phase microextraction and gas chromatography coupled with mass spectrometry to show that waggle-dancing bees produce and release two alkanes, tricosane and pentacosane, and two alkenes, Z-(9)-tricosene and Z-(9)-pentacosene, onto their abdomens and into the air. Nondancing foragers returning from the same food source produce these substances in only minute quantities. Injection of the scent significantly affects worker behavior by increasing the number of bees that exit the hive. The results of this study suggest that these compounds are semiochemicals involved in worker recruitment. By showing that honey bee waggle dancers produce and release behaviorally active chemicals, this study reveals a new dimension in the organization of honey bee foraging.

Comparative testing for the identification of skin-sensitizing potentials of nonionic sugar lipid surfactants.

The Local Lymph Node Assay (LLNA) is the preferred test for the identification of skin-sensitizing potentials of chemicals in Europe and is also the first choice method within REACH. In the formal validation, only a very few surfactant chemicals were evaluated and SDS was identified as a false positive. In this study, 10 nonionic sugar lipid surfactants were tested in an LLNA, guinea pig maximization test (GPMT) and human repeated insult patch test. Of the 10 surfactants tested in the LLNA, 5 showed stimulation indices above 3.0. Three of five positive reactions were concomitant with signs of skin irritation indicated by an increase in ear thickness. In the GPMT, all test products were classified as nonsensitizers. In human volunteers, no skin reactions suggestive of sensitization were reported. In conclusion, these results are indicative of the LLNA overestimating sensitization potentials for this category of chemicals. This may in part be due to irritant effects generated by these surfactants. Until suitable nonanimal alternative tests obtain regulatory acceptance, use of other tests, e.g. GPMTs, may in cases be justified. Results such as these need be taken into account when developing nonanimal alternative methods to ensure reliable data sets for method validation purposes.

Application of a weight of evidence approach to assessing discordant sensitisation datasets: implications for REACH.

The local lymph node assay (LLNA) is the assay of choice in European regulatory toxicology. As with other toxicology/sensitisation assays, it has a potential for false results, the anionic surfactant sodium lauryl sulphate (SLS) representing a classic example. In the work reported here, examples of false positives in the LLNA are compared to published "benchmarks" such as SLS. Clear false positives (e.g. oleic acid) are also contrasted with examples where data interpretation is more challenging. As the LLNA will be applicable to >30,000 chemicals under REACH, and in the light of animal welfare considerations to do no more than the absolute minimum of animal testing, results from a single LLNA often represent the only available data on sensitisation. This reinforces the need to ensure data from this assay are interpreted intelligently, using scientific analysis of results and considering the weight of evidence, before decisions are made on which substances should be classified as representing a skin sensitisation hazard. In chemical classes where the LLNA has been shown to be an inappropriate assay other standardised methods (e.g. the Buehler or Magnusson and Kligman guinea pig tests [OECD 406]) should be employed as the first choice assays.

Metabolic Activation of PCBs to Carcinogens in Vivo - A Review.

Many higher-chlorinated biphenyls, persistent and predominant in foods, are active as promoters in hepatocarcinogenesis. Lower-chlorinated biphenyls, predominating in indoor and outdoor air, are more readily metabolized and therefore shorter lived, 'episodic' contaminants. Thus inhalation of such lower chlorinated biphenyls may expose humans to reactive, possibly genotoxic/carcinogenic intermediates. Lower chlorinated biphenyls may be metabolized via arene-oxides to mono- and dihydroxylated intermediates and further to (semi)quinones, highly reactive intermediates. Covalently bound lower chlorinated biphenyls have been detected in rodent tissues in vivo. We recently showed using the modified Solt-Farber foci assay that several mono- to tetrachlorinated biphenyls have initiating activity in the livers of rats. In a follow-up study of PCB3 (4-chlorobiphenyl) metabolites only one monohydroxy- and one quinoid- metabolite showed initiating activity, indicating that the metabolic activation of PCB3 proceeds via hydroxylation and oxidation to the 3,4-quinone, the ultimate carcinogen. Since cancer initiation is based on genotoxic event(s), we hypothesized that PCB3 and/or its metabolite(s) are mutagenic in rat liver in vivo. To investigate this, BigBlue® rats, transgenic for the lacI reporter gene, were exposed to PCB3 and 4-hydroxy-PCB3 (4-HO-PCB3). In male rats the mutant frequency (MF) of lac I in the liver was significantly elevated and the mutation spectrum differed significantly from the control. 4-HO-PCB3 caused a non-significant (p = 0.115) doubling of the MF compared to the control. These studies prove that lower halogenated biphenyls may be metabolically activated in vivo to genotoxic and initiating intermediates.

Mutagenic potential of the isoflavone irilone in cultured V79 cells.

After consumption of red clover-based dietary supplements, plasma concentrations of the isoflavone irilone (IRI) equal that of the well-investigated daidzein. Since some isoflavones are genotoxic, the potential of IRI to induce mutations was investigated. Gene mutations were determined by hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay and sequencing of mutant cDNA, chromosome and genome mutations by micronucleus assay complemented by immunochemical staining of centromere proteins and microtubules in cultured V79 cells. Cell proliferation was monitored by electronic cell counting, flow cytometry and fluorescence microscopy. IRI did not affect the mutant frequency in the Hprt locus but altered the mutation spectrum by increasing the proportion of deletions and decreasing that of base pair substitutions. Induction of chromosome mutations was supported by a slight but significant increase in the number of micronucleated cells containing chromosomal fragments despite activation of three cell cycle checkpoints possibly interfering with micronuclei formation. Moreover, IRI exhibited a strong aneugenic potential characterized by disrupted mitotic spindles, mitotic arrest, and asymmetrical cell divisions leading to chromosome loss, nuclear fragmentation as well as mitotic catastrophe. Thus, IRI might be another isoflavone to be taken into account in safety assessment of dietary supplements.

Data in support of the mutagenic potential of the isoflavone irilone in cultured V79 cells.

The isoflavone irilone is found in human plasma after ingestion of red clover-based dietary supplements, but information allowing safety assessment is rare. Here, data in support of the mutagenic potential of irilone in cultured V79 cells [1] are presented. These data include (i) a quantitative assessment of irilone in the culture medium during the cell culture experiments, (ii) changes in the mutation spectrum in cDNA of the hypoxanthine-guanine phosphoribosyltransferase locus of irilone-treated V79 cells, (iii) occurrence of karyorrhexis and apoptosis as well as (iv) number of micronucleated cells containing whole chromosomes or chromosomal fragments. Also exemplary micrographs, used for the fluorescence microscopic assessment of (iii) and (iv) are presented.

4-monochlorobiphenyl (PCB3) induces mutations in the livers of transgenic Fisher 344 rats.

4-monochlorobiphenyl (PCB3) is found in small amounts in commercial PCB mixtures, indoor and outdoor air, and in food. In contrast to highly chlorinated congeners that are more resistant to metabolic attack, PCB3 is more readily converted by xenobiotic-metabolizing enzymes to monohydroxy-PCBs and further to dihydroxy-metabolites, which can be oxidized to quinones. Our recent studies demonstrated the initiating action of PCB3 in the livers of male rats. Therefore we hypothesized that PCB3 and/or its metabolite(s) are mutagenic in rat livers in vivo. To investigate the mutagenicity and the types of mutations generated by PCB3, male Fischer 344 BigBlue rats, transgenic for the lacI gene, were injected intraperitoneally with PCB3 (600 micromol/kg), 4-hydroxy-PCB3 (4-HO-PCB3, 400 micromol/kg), 3-methylcholanthrene (3-MC, 300 micromol/kg, positive control) and corn oil (negative control) once per week, for 4 weeks. Animals were killed 17 days after the last injection and the mutant frequency of the liver lacI gene determined. 3-MC induced a 4-fold increase of the mutant frequency of the lacI gene in the liver. The mutant frequency in PCB3-treated animals was also significantly elevated. In contrast, 4-HO-PCB3 induced a non-significant doubling of the mutant frequency. The mutation spectrum of solvent control mutants was characterized by transitions, whereas in 3-MC-animals, transversion and frameshift mutations predominated. The PCB3-induced mutation spectrum was similar to that of the 3-MC-induced mutants. In contrast, the mutation spectrum of the 4-HO-PCB3 group hardly differed from that of the control animals. This study demonstrates for the first time the mutagenicity of a PCB in vivo.