Maria João Gouveia

Schistosome and liver fluke derived catechol-estrogens and helminth associated cancers

Infection with helminth parasites remains a persistent public health problem in developing countries. Three of these pathogens, the liver flukes Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, are of particular concern due to their classification as Group 1 carcinogens: infection with these worms is carcinogenic. Using liquid chromatography-mass spectrometry (LC-MS/MS) approaches, we identified steroid hormone like (e.g., oxysterol-like, catechol estrogen quinone-like, etc.) metabolites and related DNA-adducts, apparently of parasite origin, in developmental stages including eggs of S. haematobium, in urine of people with urogenital schistosomiasis, and in the adult stage of O. viverrini. Since these kinds of sterol derivatives are metabolized to active quinones that can modify DNA, which in other contexts can lead to breast and other cancers, helminth parasite associated sterols might induce tumor-like phenotypes in the target cells susceptible to helminth parasite associated cancers, i.e., urothelial cells of the bladder in the case of urogenital schistosomiasis and the bile duct epithelia or cholangiocytes, in the case of O. viverrini and C. sinensis. Indeed we postulate that helminth induced cancers originate from parasite estrogen-host epithelial/urothelial cell chromosomal DNA adducts, and here we review recent findings that support this conjecture.

Mass spectrometry techniques in the survey of steroid metabolites as potential disease biomarkers: A review

Mass spectrometric approaches have been fundamental to the identification of metabolites associated with steroid hormones, yet this topic has not been reviewed in depth in recent years. To this end, and given the increasing relevance of liquid chromatography-mass spectrometry (LC-MS) studies on steroid hormones and their metabolites, the present review addresses this subject. This review provides a timely summary of the use of various mass spectrometry-based analytical techniques during the evaluation of steroidal biomarkers in a range of human disease settings. The sensitivity and specificity of these technologies are clearly providing valuable new insights into breast cancer and cardiovascular disease. We aim to contribute to an enhanced understanding of steroid metabolism and how it can be profiled by LC-MS techniques.

Tumour-like phenotypes in urothelial cells after exposure to antigens from eggs of Schistosoma haematobium: an oestrogen-DNA adducts mediated pathway?

Chronic infection with the blood fluke, Schistosoma haematobium, is associated with squamous cell carcinoma of the bladder. Previously, it has been shown that soluble extracts of mixed sex adult S. haematobium worms (SWAP) are tumourigenic, both in vitro and in vivo. In addition, oestrogen-related molecules in SWAP of S. haematobium down-regulate oestrogen receptors (ERs) alpha and beta in oestrogen responsive cells. Moreover, schistosome oestrogens occur in sera of persons with schistosomiasis haematobia and repress transcription of ERs in urothelial cells. Given that eggs of S. haematobium are the developmental stage directly responsible for urogenital disease during schistosomiasis haematobia, we suspected that soluble antigens from S. haematobium eggs exhibit similar or more potent tumorigenic capacity. Here we investigated the tumorigenic potential of soluble egg antigens (Sh-SEA) of S. haematobium and the endocrine system in favouring parasitism by schistosomes. The findings confirmed that 6.25μg/ml of Sh-SEA was enough to stimulate cell proliferation, reduce apoptosis and increase oxidative stress of Sh-SEA-exposed urothelial cells. In addition, genotoxic effects of Sh-SEA on these cells were determined by using alkaline single-cell gel electrophoresis (Comet). Furthermore, Liquid Chromatography Diode Array Detection Electron Spray Ionisation Mass Spectrometry indicated the presence of catechol-oestrogens in S. haematobium SEA. A prospective oestrogen-DNA adduct mediated pathway in S. haematobium egg induced bladder cancer is also discussed.

Urinary estrogen metabolites and self-reported infertility in women infected with Schistosoma haematobium

Background Schistosomiasis is a neglected tropical disease, endemic in 76 countries, that afflicts more than 240 million people. The impact of schistosomiasis on infertility may be underestimated according to recent literature. Extracts of Schistosoma haematobium include estrogen-like metabolites termed catechol-estrogens that down regulate estrogen receptors alpha and beta in estrogen responsive cells. In addition, schistosome derived catechol-estrogens induce genotoxicity that result in estrogen-DNA adducts. These catechol estrogens and the catechol-estrogen-DNA adducts can be isolated from sera of people infected with S. haematobium. The aim of this study was to study infertility in females infected with S. haematobium and its association with the presence of schistosome-derived catechol-estrogens. Methodology/Principal Findings A cross-sectional study was undertaken of female residents of a region in Bengo province, Angola, endemic for schistosomiasis haematobia. Ninety-three women and girls, aged from two (parents interviewed) to 94 years were interviewed on present and previous urinary, urogenital and gynecological symptoms and complaints. Urine was collected from the participants for egg-based parasitological assessment of schistosome infection, and for liquid chromatography diode array detection electron spray ionization mass spectrometry (LC/UV-DAD/ESI-MSn) to investigate estrogen metabolites in the urine. Novel estrogen-like metabolites, potentially of schistosome origin, were detected in the urine of participants who were positive for eggs of S. haematobium, but not detected in urines negative for S. haematobium eggs. The catechol-estrogens/ DNA adducts were significantly associated with schistosomiasis (OR 3.35; 95% CI 2.32–4.84; P≤0.001). In addition, presence of these metabolites was positively associated with infertility (OR 4.33; 95% CI 1.13–16.70; P≤0.05). Conclusions/Significance Estrogen metabolites occur widely in diverse metabolic pathways. In view of the statistically significant association between catechol-estrogens/ DNA adducts and self-reported infertility, we propose that an estrogen-DNA adduct mediated pathway in S. haematobium-induced ovarian hormonal deregulation could be involved. In addition, the catechol-estrogens/ DNA adducts described here represent potential biomarkers for schistosomiasis haematobia.

Carcinogenic liver fluke Opisthorchis viverrini oxysterols detected by LC-MS/MS survey of soluble fraction parasite extract.

Liquid chromatography in tandem mass spectrometry (LC-MS/MS) has emerged as an informative tool to investigate oxysterols (oxidized derivatives of cholesterol) in helminth parasite associated cancers. Here, we used LC-MS/MS to investigate in soluble extracts of the adult developmental stage of Opisthorchis viverrini from experimentally infected hamsters. Using comparisons with known bile acids and the metabolites of estrogens, the LC-MS data indicated the existence of novel oxysterol derivatives in O. viverrini. Most of these derivatives were ramified at C-17, in similar fashion to bile acids and their conjugated salts. Several were compatible with the presence of an estrogen core, and/or hydroxylation of the steroid aromatic ring A, hydroxylation of both C-2 and C-3 of the steroid ring and further oxidation into an estradiol-2,3-quinone.

Praziquantel for Schistosomiasis: Single-Drug Metabolism Revisited, Mode of Action, and Resistance

ABSTRACT Schistosomiasis, a major neglected tropical disease, affects more than 250 million people worldwide. Treatment of schistosomiasis has relied on the anthelmintic drug praziquantel (PZQ) for more than a generation. PZQ is the drug of choice for the treatment of schistosomiasis; it is effective against all major forms of schistosomiasis, although it is less active against juvenile than mature parasites. A pyrazino-isoquinoline derivative, PZQ is not considered to be toxic and generally causes few or transient, mild side effects. Increasingly, mass drug administration targeting populations in sub-Saharan Africa where schistosomiasis is endemic has led to the appearance of reduced efficacy of PZQ, which portends the selection of drug-resistant forms of these pathogens. The synthesis of improved derivatives of PZQ is attracting attention, e.g., in the (i) synthesis of drug analogues, (ii) rational design of pharmacophores, and (iii) discovery of new compounds from large-scale screening programs. This article reviews reports from the 1970s to the present on the metabolism and mechanism of action of PZQ and its derivatives against schistosomes.

The role of estradiol metabolism in urogenital schistosomiasis-induced bladder cancer

Urogenital schistosomiasis is a neglected tropical disease that can lead to bladder cancer. How urogenital schistosomiasis induces carcinogenesis remains unclear, although there is evidence that the human blood fluke Schistosoma haematobium, the infectious agent of urogenital schistosomiasis, releases estradiol-like metabolites. These kind of compounds have been implicated in other cancers. Aiming for enhanced understanding of the pathogenesis of the urogenital schistosomiasis-induced bladder cancer, here we review, interpret, and discuss findings of estradiol-like metabolites detected in both the parasite and in the human urine during urogenital schistosomiasis. Moreover, we predict pathways and enzymes that are involved in the production of these metabolites emphasizing their potential effects on the dysregulation of the tumor suppressor gene p53 expression during urogenital schistosomiasis. Enhanced understanding of these potential carcinogens may not only shed light on urogenital schistosomiasis-induced neoplasia of the bladder, but would also facilitate development of interventions and biomarkers for this and other infection-associated cancers at large.

Drug Repurposing for Schistosomiasis: Combinations of Drugs or Biomolecules

Schistosomiasis is a major neglected tropical disease. Control of schistosomiasis currently relies on a single drug, praziquantel, and despite its efficacy against the all schistosome species that parasitize humans, it displays some problematic drawbacks and alone is ineffective in counteracting adverse pathologies associated with infection. Moreover, due to the development of the potential emergence of PZQ-resistant strains, the search for additional or alternative antischistosomal drugs have become a public health priority. The current drug discovery for schistosomiasis has been slow and uninspiring. By contrast, repurposing of existing approved drugs may offer a safe, rapid and cost-effective alternative. Combined treatment with PZQ and other drugs with different mode of action, i.e., antimalarials, shows promise results. In addition, a combination of anthelminthic drugs with antioxidant might be advantageous for modulating oxidative processes associated with schistosomiasis. Herein, we review studies dealing with combination therapies that involve PZQ and other anthelminthic drugs and/or antioxidant agents in treatment of schistosomiasis. Whereas PZQ combined with antioxidant agents might or might not interfere with anthelminthic efficacy, combinations may nonetheless ameliorate tissue damage and infection-associated complications. In fact, alone or combine with other drugs, antioxidants might be a valuable adjuvant to reduce morbidity and mortality of schistosomiasis. Therefore, attempting new combinations of anthelmintic drugs with other biomolecules such as antioxidants provides new avenues for discovery of alternatives to PZQ.

Infection with Opisthorchis felineus induces intraepithelial neoplasia of the biliary tract in a rodent model.

The liver fluke Opisthorchis felineus is a member of the triad of epidemiologically relevant species of the trematode family Opisthorchiidae, and the causative agent of opisthorchiasis felinea over an extensive range that spans regions of Eurasia. The International Agency for Research on Cancer classifies the infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis as group 1 agents and a major risk factor for cholangiocarcinoma. However, the carcinogenic potential of the infection with O. felineus is less clear. Here, we present findings that support the inclusion of O. felineus in the Group 1 list of biological carcinogens. Two discrete lines of evidence support the notion that infection with this liver fluke is carcinogenic. First, novel oxysterol-like metabolites detected by liquid chromatography-mass spectroscopy in the egg and adult developmental stages of O. felineus, and in bile, sera, and urine of liver fluke-infected hamsters exhibited marked similarity to oxysterol-like molecules known from O. viverrini. Numerous oxysterols and related DNA-adducts detected in the liver fluke eggs and in bile from infected hamsters suggested that infection-associated oxysterols induced chromosomal lesions in host cells. Second, histological analysis of liver sections from hamsters infected with O. felineus confirmed portal area enlargement, inflammation with severe periductal fibrosis and changes in the epithelium of the biliary tract characterized as biliary intraepithelial neoplasia, BilIN. The consonance of these biochemical and histopathological changes revealed that O. felineus infection in this rodent model induced precancerous lesions conducive to malignancy.

Amino Acids in the Development of Prodrugs

Although drugs currently used for the various types of diseases (e.g., antiparasitic, antiviral, antibacterial, etc.) are effective, they present several undesirable pharmacological and pharmaceutical properties. Most of the drugs have low bioavailability, lack of sensitivity, and do not target only the damaged cells, thus also affecting normal cells. Moreover, there is the risk of developing resistance against drugs upon chronic treatment. Consequently, their potential clinical applications might be limited and therefore, it is mandatory to find strategies that improve those properties of therapeutic agents. The development of prodrugs using amino acids as moieties has resulted in improvements in several properties, namely increased bioavailability, decreased toxicity of the parent drug, accurate delivery to target tissues or organs, and prevention of fast metabolism. Herein, we provide an overview of models currently in use of prodrug design with amino acids. Furthermore, we review the challenges related to the permeability of poorly absorbed drugs and transport and deliver on target organs.