Toxicological evaluation of inhalation exposure to benzene and toluene in a raptorial bird, the American kestrel, falco sparverius

Benzene and toluene are representative volatile organic compounds (VOCs) released during production, storage, and transportation associated with the oil and gas industry. Benzene and toluene are chemicals of concern because they are released in greater and possibly more biologically significant concentrations than other compounds. Most studies of air pollution in high oil and gas activity areas have neglected to consider risks to top-level predators. Birds can be used as highly sensitive monitors of air quality. Since the avian respiratory tract is physiologically different from a rodent respiratory tract, effects of gases cannot be safely extrapolated from rodent studies. I hypothesized that benzene, being haematotoxic and immunotoxic, along with the neurological and possible endocrine disrupting effects of toluene would be more toxic in birds than in mammals. After two summers of experimental exposure of wild and captive American kestrels to high (10ppm and 80ppm) or environmentally relevant (0.1ppm and 0.8ppm) levels of benzene and toluene, respectively, altered immune, haematopoeitic, behavioural, and endocrine responses characteristic in mammals, were evident in the kestrels.There was a decreased cell mediated immune response as measured by delayed type hypersensitivity tests in all exposed birds (p = 0.028, 0.004). An increase in humoral immunity as compared to control individuals (p = 0.041, 0.031) was also apparent in both dose groups. Plasma retinol levels were decreased in 2005 and 2006 high dose individuals (p = 0.008, 0.048). The majority of haematopoeitic effects involved the erythroid lineage in the bone marrow and the polychromatophilic erythrocytes systemically. There were no significantly adverse responses in the bone marrow with regards to the granuloid lineage but systemically there was a prominent eosinophilia (p = 0.045) and basophilia (p = 0.006) in low exposure groups. The loss of communication between polychromatophilic erythrocytes in the post-mitotic pool within the bone marrow and the peripheral blood was present in low and high exposure individuals compared to control birds (p = 0.013, 0.402, 0.974). The number of polychromatophils in the circulation of low dose group individuals was decreased compared to control birds (p = 0.029). This may be a function of toluene’s inability to inhibit biotransformation enzymes at low concentrations leading to blood cell targeting by benzene’s increased phenolic metabolite production. This theory is corroborated by the possible decreased benzene metabolism and increased toluene distribution manifesting as increased aggressive responses such as wing beating and vocalization time in the high dose group (p = 0.025, 0.086). The work here has shown American kestrels are sensitive to the air contaminants, benzene and toluene. The present study illustrates the need for reference concentrations for airborne pollutants that are calculated based on data measuring sensitive endpoints specific for avian models. Future studies should evaluate immune, haematopoeitic, and behavioural endpoints, as well as develop more sensitive isoform specific enzyme activity assays to further determine the susceptibility of birds to inhaled toxicants

Amifostine (WR-2721) selective protection against melphalan genotoxicity

Amifostine (WR-2721) is an aminothiol compound dephosphorylated at the tissue site by alkaline phosphatase to the active metabolite, which is able to inactivate electrophilic substances and scavenge free radicals. Amifostine effects against melphalan-induced DNA strand breaks were studied in normal human white blood cells (WBC) and K562 leukemic cells using the single cell gel electrophoresis (SCGE) or Comet assay, a reported method for measuring DNA damage in individual cells. Prior to treatment (1 h, 37 degrees C) with increasing doses of melphalan, with or without S9, the cells were treated (15 min, 37 degrees C) with a control medium or amifostine (3 mg/ml). Treatment of normal and leukemic cells with melphalan induced a dose-dependent 'comet formation'. Melphalan-induced DNA damage follows a normal distribution in WBC. On the other hand, in K562, a significant proportion of undamaged cells remains even with doses at which mean DNA damage is serious. Pretreatment with WR-2721 protects WBC, but not K562, against the genotoxic effect of melphalan, Amifostine might even strengthen the action of the antiblastic drug against K562 cells. S9 addition appears to enhance melphalan effectiveness. SCGE appears as a suitable primary screening method for in vitro and in vivo studies on drug-DNA interactions and their modulations by endogenous/exogenous factors

Leukemia treatment: studies exploring bone marrow microenvironment, drug resistance and cannabidiol

The results described in this thesis highlight the role of human regenerative associated endothelial cells (hRECs) in the bone marrow microenvironment and hematopoiesis. By being resistant to 5-FU therapy, hRECs remain active in secreting factors enabling enhanced expansion of precursor HSC. A second part of this thesis provides novel insights into the role of specific influx and efflux transporters implicated in the efficacy/resistance of MTX and Ara-C. Finally, a third part described the results of in vitro experimental anti-leukemia therapeutics with cannabidiol, revealing double-edged effects: anti-leukemic effects at high doses of CBD and cell growth stimulatory effects at low doses of CBD. Together, these results may contribute to improve anti-leukemic chemotherapy efficacy and minimize toxicity

Chemotherapy induces a genotoxic bystander effect in cell lines from the human bone marrow; evaluation of the role of the redox microenvironment

Modern chemotherapeutic regimens for adult and childhood leukaemia rely on the use of a combination of various cytotoxic agents. Unfortunately, haematopoietic stem cell transplant, a last resort treatment for haematologic malignancies requires high dose of chemotherapy prior to transplant. Despite advances in cancer chemotherapy, occurrence of secondary malignancies such as donor cell leukaemia (DCL) persist due to the random targets of actively dividing cells by chemotherapy. DCL is a rare condition whereby infused donor cells become toxic in the patient, despite the donor apparently remaining healthy. To date, the cause of DCL is unknown, therefore this thesis sought to explore the role of chemotherapy-induced bystander effect (CIBE) in DCL.To investigate CIBE, a co-culture bystander model was developed, promoting cell-to-cell communication in vitro and permitting isolation of the bystander cells for cytotoxicity and genotoxicity assessment post exposure to chemotherapeutic agents. For the first time, a panel of 22 chemotherapeutic agents were screened for CIBE and were found to promote genotoxicity in bystander cells. Four drugs (chlorambucil, carmustine, etoposide and mitoxantrone) later studied in detail suggested mitoxantrone induced mainly aneugenic events, but a mixed mode of function for the other three drugs swaying towards aneugenicity in the pancentromeric labelling of micronucleus assay. Interestingly, these analyses demonstrated a novel finding that the donor or bystander cells or both, may play an important role in the occurrence of CIBE, as responses within the TK6, AHH-1 and Kasumi-1 bystander cells differed throughout the research.The role of the tumour microenvironment in potentiating CIBE also appears important as genotoxicity peaked at day 3 and continued up to 5 days post chemotherapy exposure. Use of the comet assay did not demonstrate significant DNA fragmentation but addition of hOGG1 and Fpg endonucleases suggested a role for redox disruption and generation of reactive oxygen species (ROS). Investigation of the redox status showed an increase in ROS in the bone marrow microenvironment (HS-5 cells), but show little evidence of ROS in bystander cells, despite the increased oxidative damage in the comet assay. The Agilent Seahorse extracellular flux (XF) mitochondrial stress test supported redox disruption in the HS-5 cells, but analyses of antioxidant expression in both compartments showed limited evidence of any response (either increased or decreased).Collectively this research has demonstrated the capacity for chemotherapeutic agents to induce a bystander effect with the capacity to promote development of a mutated clone within a supportive tumour microenvironment. Currently the mechanism of this bystander effect is unknown, with redox disruption unlikely to play a major direct role. However, given the understanding of ROS in cellular signalling, and the observation of increased oxidative damage, this may promote cell survival within a genotoxic environment, which warrants further investigation. The data notionally could support a role for CIBE in the development of DCL, but also raises important questions in the wider applications of genotoxicity testing and models used as hitherto, the focus has been on outcomes from direct exposure, with monocultures used as the mainstay. This raises important implications for the assessment of human risk and support the idea of more complex multicellular and 3-dimensional study models

Studies on the behaviour of human pancreatic carcinoma cell lines, with particular reference to stem cells

PhDStem cell therapy represents a potential -cell replacement strategy in diabetes and a pancreatic cancer (PC) stem cell is an attractive therapeutic target in this dire disease. However, a normal stem cell remains unidentified and proposed malignant stem cell markers await verification. Using human PC cell lines as an in vitro tumour model and human archival tissue, and proposed stem cell markers identified in other normal tissues and tumours, we attempted to identify a PC stem cell. These markers included high aldehyde dehydrogenase activity, the Side Population (SP) phenotype, and the cell surface markers CD44 and CD133. These markers were evaluated by flow cytometry, immunohistochemistry (IHC) and reverse-transcriptase PCR. Positive populations were isolated from cell lines using fluorescence activated cell sorting (FACS) and subjected to in vitro colony-forming assays to assess their relative proliferative capacities. The presence of a differentiation hierarchy was investigated in one line to test the assumptions of the tumour model. No single marker or combination thereof consistently identified a highly clonogenic fraction, though some differences in clonogenicity were seen. Furthermore, the dye Hoechst 33342 was found to inhibit proliferation, potentially inducing artefactual differences in clonogenicity between SP and non-SP cells. Recently, expression of trefoil factor family peptide (TFF) 3, a gastrointestinal motogen, was reported in human islets of Langerhans. Furthermore, this peptide was reported as a -cell mitogen in vitro, and a potential agent of functional -cell production. We employed isotopic in situ hybridization and IHC to evaluate TFF expression in normal human pancreas, chronic pancreatitis and PC. Expression of all three TFFs was increased in inflammation and cancer, and TFF3 mRNA was occasionally detected in islets. The motogenic activity of TFF peptides was investigated using in vitro wound healing and invasion assays on human PC lines and primary stellate cell cultures. TFF2 was seen to enhance migration in PC lines at high concentrations

Histopathological changes in male wistar rats maintained on a water-based sutherlandia frutescens extract

In this study a standardized 46 week chronic drinking water toxicity protocol was used to elucidate the toxic potential of Sutherlandia frutescens (S. frutescens) using histopathologic, morphometric and transmission electron microscopic analysis. The histopathologic changes in the duodenum, heart, kidney, liver, lung, pancreas and spleen of male Wistar rats were evaluated. Fifty-four rats were randomly divided into four groups: Group 1 – Normal diet control (ND control), n=7, Group 2 – Normal diet + plant extract (ND + p), n=9, Group 3 – High fat diet control (HFD control), n=19Group 4 – High fat diet + p (HFD + p), n=19In the high fat group male Wistar rats were fed ±55 g/day of a specialised high fat diet over a 46 week period to induce obesity and an insulin resistant state. The treatment groups (groups 2 and 4) received a dose concentration of a tea extract of the S. frutescens plant in their drinking water daily. This study showed that the consumption of S. frutescens significantly reduces weight gain in male Wistar rats on a chronic high fat diet (p≤0.001 vs. HFD control group). S. frutescens appears to propagate periportal and centrilobular glycogen storage in rat hepatocytes in the experimental groups as exemplified by a significantly (p≤0.0001 vs. control groups) increased incidences of Periodic Acid Schiff (PAS) positive staining S. frutescens also reduced intracellular lipid accumulation as made evident by the significantly lower incidence of epicardial adipose tissue (EAT), hepatic steatosis and pancreatic interstitial fat. Obesity was associated with increased fibrotic lesions such as myocardial perivascular fibrosis, centrilobular hepatic fibrosis and pancreatic periductal fibrosis. Obesity associated hypertension contributed to the widespread and significant increase in the average lesion severity of arterial congestion in all organs in the HFD control group. Pulmonary infection was equally prevalent in all rats. Despite the complex histopathology in all groups, differences in the control groups, such as, the presence of a conservative polymorphonuclear leukocyte (PMNL) infiltration, substantial intra-alveolar oedema and focal arterial wall hypertrophy in the control groups was highly suggestive of Sendai viral infection. However histopathologic evidence, in the treatment groups, suggested chronic recurrent viral infection with superimposed Mycoplasma pulmonis (M. pulmonis) bacterial infection. The impact of advanced suppurative pulmonary infection was widespread and exemplified by increased lesion incidences of spontaneous murine progressive cardiomyopathy (MCP) and spontaneous chronic progressive nephropathy (CPN) among others. In conclusion S. frutescens administered for 46 weeks to male Wistar rats significantly lowered intracellular lipid accumulation and obesity associated myocardial, renal, hepatobiliary, pulmonary and pancreatic histopathology. Moreover, duodenal, cardiovascular, hepatobiliary, pulmonary, renal, pancreatic and splenic tissue did not show histopathologic evidence of direct plant extract associated toxicity or carcinogenicity

Arüülsüsivesinike retseptor hiire munasarja granuloosarakkudes

Väitekirja elektrooniline versioon ei sisalda publikatsiooneOn teada, et keskkonnas levinud kemikaalid, sealhulgas dioksiinid, mõjutavad oluliselt organismi talitlust, põhjustades näiteks vähki ning häireid immuun- ja reproduktiivsüsteemis. Paljud neist ühenditest on ligandiks arüülsüsivesinike retseptorile (AHR). Ligandi poolt aktiveerituna liigub AHR raku tsütoplasmast tuuma, kus mõjutab erinevate geenide avaldumist. Nende hulka kuuluvad kemikaale lagundavate ensüümide geenid, aga ka hulganisti rakkude normaalseks talitluseks vajalikke geene. Mitmed teaduspublikatsioonid on näidanud, et erinevates vähitüüpides on AHR-i tase ning aktiivsus väga kõrge ning see soodustab kasvaja arengut, mistõttu on AHR huvipakkuvaks sihtmärgiks vähiuuringutes. Seejuures on oluline märkida, et peamiselt keskendutakse just AHR-i aktiivsuse moduleerimisele ning selle enda regulatsioonimehhanismid on jäänud pigem tagaplaanile. Ahr-i roll reproduktiivsüsteemis on seotud eelkõige folliikuleid stimuleeriva hormooni retseptori (Fshr) ja aromataasi positiivse regulatsiooniga. Need geenid on olulised munasarjas asuvate granuloosarakkude funktsioneerimiseks, toetades seeläbi munaraku küpsemist ning ovulatsiooni. Samuti on oluline märkida, et Ahr-i enda ekspressioon muutub reproduktiivtsükli jooksul. Käesoleva doktoritöö eesmärgiks oli selgitada Ahr-i regulatsioonimehhanisme hiire munasarja granuloosarakkudes enne ja pärast ovulatsiooni stimuleerimist ning tuvastada, kuidas täpsemalt mõjutab Ahr Fshr-i ekspressiooni. Selgus, et granuloosarakkude küpsemise käigus suureneb Ahr-i ekspressioon oluliselt ning see on põhjustatud proteiinkinaas A (PKA) signaliseerimise lühiaegsest nõrgenemisest. Lisaks leiti, et Ahr-i ekspressioon väheneb oluliselt ovulatsiooni indutseerimise järgselt ning see on tingitud just PKA aktiivsusest. Viimaks tehti kindlaks uudne DNA järjestus, millele Ahr on võimeline seonduma ning Fshr-i ekspressiooni reguleerima.Environmental chemicals, including dioxins, affect the homeostasis of organism resulting in cancer and disturbances in the immune and reproductive system. Many of these chemicals act as ligands for aryl hydrocarbon receptor (AHR). Upon ligand binding, AHR translocates to the nucleus and modulates the expression of a variety of genes. Among these are genes responsible for the degradation of AHR ligands, but also genes necessary for normal functioning of cells. Several publications have shown high AHR expression and activity in cancers. Thus, AHR is a promising target in cancer research. However, the majority of attention has been focused on its activity modulation and the mechanisms governing AHR expression have stayed rather unclear. The role of AHR in reproduction is mainly associated with its positive effect on follicle stimulating hormone receptor (Fshr) and aromatase. These two genes are crucial for the proper functioning of granulosa cells, which support the maturation and ovulation of oocyte. In addition, it has been shown that Ahr expression fluctuates during reproductive cycle. The aim of this thesis was to characterise the control of Ahr expression in mouse ovarian granulosa cells and to clarify by which mechanisms Ahr influences Fshr expression. It was uncovered that Ahr is upregulated via transient silencing of protein kinase A (PKA) signalling during maturation of granulosa cells. Following induction of ovulation, in turn, Ahr is silenced by PKA pathway. Lastly, a novel binding site for Ahr was ascertained by which Ahr regulates Fshr expression.https://www.ester.ee/record=b536898

Terve ja kahjustatud maksa regeneratsioonis ning Dupuytrenʼi kontraktuuri progressioonis osalevate rakuliste ja molekulaarsete mehhanismide uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Maks on imetaja suurim siseorgan, mis omab unikaalset taastumisvõimet, kuid pidev krooniline maksakahjustus põhjustab tema regeneratsioonivõime vähenemist, fibroosse armkoe tekkimist ja maksa funktsioonide kadumist. Sageli on lõppstaadiumis oleva kroonilise maksahaiguse ainsaks ravivõimaluseks maksasiirdamine. Leidmaks maksasiirdamisele alternatiivseid ravimeetodeid, on oluline uurida maksa regeneratsiooni ja haiguslike protsessidega seotud molekulaarseid ja rakulisi mehhanisme. Käesolevas töös uurisime, kuidas maksa aeglaselt jagunevad rakud panustavad koe taastamisse, millised rakuvälise maatriksi muudatused leiavad aset vastusena maksakahjustusele ja kuidas need võiksid mõjutada rakkude käitumist. Samuti uurisime, kas signaalirajad, mis on olulised maksafibroosis, omavad rolli ka teist tüüpi fibrootilises haiguses ja lõpuks viisime läbi suuremahulise kemikaalide skriinimise, leidmaks AKT1-PDPK1 interaktsiooni inhibiitorit, mis vähendaks kasvajate-ja fibroosiseoselise AKT1 aktivatsiooni. Töö tulemusena leidsime, et maksa aeglaselt jagunevad rakud on unipotentsed sapijuharakud, mis aktiveeruvad ainult kroonilise kahjustuse korral. Samuti selgus, et maksa rakuvälise maatriksi muutused sõltuvad kahjustuse tüübist ning erinevad maatriksi komponendid omavad hepatotsüütide ja sapijuharakkude proliferatsioonile erinevat mõju. Uurides fibrootilisi protsesse Dupuytren´i kontraktuuri koes, avastasime, et haige koe erinevad komponendid sünteesivad erinevaid fibroosi ja proliferatsiooni soodustavaid molekule, moodustades haiguse arenguks sobiliku keskonna. Töö viimases osas kirjeldasime AKT1-PDPK1 interaktsiooni inhibiitori, väikesemolekulaarse ühendi NSC156529, identifitseerimise protsessi. NSC156529 vähendas AKT valgu ja tema sihtmärkvalkude aktiivsust ning pidurdas rakkude kasvu in vitro ja tuumori kasvu in vivo. Seega on NSC156529 sobiv alusmolekul uudsete ravimite väljatöötamiseks selliste haiguste nagu kasvajad ning fibroos vastu, mille puhul AKT signaaliraja aktiivsus on kõrgenenud.The liver is the largest mammalian visceral organ with a remarkable regenerative capacity. However, in case of persistent chronic liver injury, this regenerative potential declines, the parenchyma becomes fibrotic, and finally, the liver is not able to exert its biological functions anymore. Since liver transplantation is often the only treatment option for end-stage chronic liver diseases, development of alternative therapies has become increasingly important. In order to generate new treatments, it is necessary to understand the cellular and molecular mechanisms that control liver regeneration and disease progression. The first objective of this thesis was to determine the role of slowly-cycling liver cells in liver regeneration. Secondly, we studied the changes in extracellular matrix (ECM) that occur in response to different liver injuries, and the effect of these alterations on the regenerative properties of liver cells. We also studied, how the signaling pathways that are associated with liver fibrosis, are involved in another type of fibrotic disease–Dupuytren’s contracture (DC). And finally we conducted a large-scale chemical screening to find inhibitors for AKT1-PDPK1interaction that would suppress AKT pathway, which is aberrantly activated in many cancers and fibrotic diseases. We discovered that slowly-cycling liver cells are unipotent biliary cells which are activated and take part in liver regeneration only in response to chronic liver injury. The changes in ECM appear to be dependent on the type of injury, and the hepatocytes and cholangiocytes have different proliferative activity on different matrix components. When studying fibrotic processes in DC we found that different compartments in DC tissue synthesize distinct fibrosis-and proliferation-inducing molecules, which synergize in forming a supportive microinvironment for disease progression. In the last part of this work we described the identification process of NSC156529–the novel inhibitor of AKT1-PDPK1 interaction. NSC156529 is a small molecule compound that inhibited AKT pathway and restricted cell growth in vitro and tumor growth in vivo. We proposed that NSC156529 is a new strong candidate for the development of novel anti-tumor therapeutics

Senescence

The book "Senescence" is aimed to describe all the phenomena related to aging and senescence of all forms of life on Earth, i.e. plants, animals and the human beings. The book contains 36 carefully reviewed chapters written by different authors, aiming to describe the aging and senescent changes of living creatures, i.e. plants and animals