Specific Increase in MDR1 Mediated Drug-Efflux in Human Brain Endothelial Cells following Co-Exposure to HIV-1 and Saquinavir

Persistence of HIV-1 reservoirs within the Central Nervous System (CNS) remains a significant challenge to the efficacy of potent anti-HIV-1 drugs. The primary human Brain Microvascular Endothelial Cells (HBMVEC) constitutes the Blood Brain Barrier (BBB) which interferes with anti-HIV drug delivery into the CNS. The ATP binding cassette (ABC) transporters expressed on HBMVEC can efflux HIV-1 protease inhibitors (HPI), enabling the persistence of HIV-1 in CNS. Constitutive low level expression of several ABC-transporters, such as MDR1 (a.k.a. P-gp) and MRPs are documented in HBMVEC. Although it is recognized that inflammatory cytokines and exposure to xenobiotic drug substrates (e.g HPI) can augment the expression of these transporters, it is not known whether concomitant exposure to virus and anti-retroviral drugs can increase drug-efflux functions in HBMVEC. Our in vitro studies showed that exposure of HBMVEC to HIV-1 significantly up-regulates both MDR1 gene expression and protein levels; however, no significant increases in either MRP-1 or MRP-2 were observed. Furthermore, calcein-AM dye-efflux assays using HBMVEC showed that, compared to virus exposure alone, the MDR1 mediated drug-efflux function was significantly induced following concomitant exposure to both HIV-1 and saquinavir (SQV). This increase in MDR1 mediated drug-efflux was further substantiated via increased intracellular retention of radiolabeled [3H-] SQV. The crucial role of MDR1 in 3H-SQV efflux from HBMVEC was further confirmed by using both a MDR1 specific blocker (PSC-833) and MDR1 specific siRNAs. Therefore, MDR1 specific drug-efflux function increases in HBMVEC following co-exposure to HIV-1 and SQV which can reduce the penetration of HPIs into the infected brain reservoirs of HIV-1. A targeted suppression of MDR1 in the BBB may thus provide a novel strategy to suppress residual viral replication in the CNS, by augmenting the therapeutic efficacy of HAART drugs.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

Effect of reaction mechanism on precursor exposure time in atomic layer deposition of silicon oxide and silicon nitride

Atomic layer deposition (ALD) of highly conformal, silicon-based dielectric thin films has become necessary because of the continuing decrease in feature size in microelectronic devices. The ALD of oxides and nitrides is usually thought to be mechanistically similar, but plasma-enhanced ALD of silicon nitride is found to be problematic, while that of silicon oxide is straightforward. To find why, the ALD of silicon nitride and silicon oxide dielectric films was studied by applying ab initio methods to theoretical models for proposed surface reaction mechanisms. The thermodynamic energies for the elimination of functional groups from different silicon precursors reacting with simple model molecules were calculated using density functional theory (DFT), explaining the lower reactivity of precursors toward the deposition of silicon nitride relative to silicon oxide seen in experiments, but not explaining the trends between precursors. Using more realistic cluster models of amine and hydroxyl covered surfaces, the structures and energies were calculated of reaction pathways for chemisorption of different silicon precursors via functional group elimination, with more success. DFT calculations identified the initial physisorption step as crucial toward deposition and this step was thus used to predict the ALD reactivity of a range of amino-silane precursors, yielding good agreement with experiment. The retention of hydrogen within silicon nitride films but not in silicon oxide observed in FTIR spectra was accounted for by the theoretical calculations and helped verify the application of the model.

Developmental toxicity from exposure to various forms of mercury compounds in medaka fish (Oryzias latipes) embryos.

This study examined developmental toxicity of different mercury compounds, including some used in traditional medicines. Medaka (Oryzias latipes) embryos were exposed to 0.001-10 µM concentrations of MeHg, HgCl2, α-HgS (Zhu Sha), and β-HgS (Zuotai) from stage 10 (6-7 hpf) to 10 days post fertilization (dpf). Of the forms of mercury in this study, the organic form (MeHg) proved the most toxic followed by inorganic mercury (HgCl2), both producing embryo developmental toxicity. Altered phenotypes included pericardial edema with elongated or tube heart, reduction of eye pigmentation, and failure of swim bladder inflation. Both α-HgS and β-HgS were less toxic than MeHg and HgCl2. Total RNA was extracted from survivors three days after exposure to MeHg (0.1 µM), HgCl2 (1 µM), α-HgS (10 µM), or β-HgS (10 µM) to examine toxicity-related gene expression. MeHg and HgCl2 markedly induced metallothionein (MT) and heme oxygenase-1 (Ho-1), while α-HgS and β-HgS failed to induce either gene. Chemical forms of mercury compounds proved to be a major determinant in their developmental toxicity.

Reproductive effects of endocrine disrupting chemicals, bisphenol-A and 17β-oestradiol, on Cerastoderma edule from south-west England: field study and laboratory exposure

Endocrine disruption has rarely been reported in field populations of the edible cockle and the context with the general health of the shellfish is unclear. This study examined the reproductive state of two Cerastoderma edule populations over a 6-month period to assess their reproductive condition, the incidence of intersex and presence of parasitic infection. A further seven native sites from south-west England were examined during the peak reproductive season to identify the presence of intersex within the region. Laboratory exposures of organisms collected from field populations showed a significantly female-biased sex ratio compared with controls when exposed to the endocrine disrupting chemicals, bisphenol-A (nominal concentration: 0.1 µg L−1) and 17β-oestradiol (nominal concentration: 0.1 µg L−1), but none of the chemical exposures induced intersex. Intersex was revealed in seven out of the nine native populations of C. edule sampled at peak reproductive season. The highest incidence and most severe case of intersex were reported at Lower Anderton on the River Tamer which also had a significantly female-biased sex ratio. Additionally, the dominant trematode family was the Bucephalaidae. Parasitic infection influences the maturity of C. edule by lowering both mean gonad index and condition index. These results suggest that endocrine disrupting chemicals could be contributing factors towards the development of intersex in C. edule.

Reproductive effects of endocrine disrupting chemicals, bisphenol-A and 17β-oestradiol, on Cerastoderma edule from south-west England: field study and laboratory exposure

Endocrine disruption has rarely been reported in field populations of the edible cockle and the context with the general health of the shellfish is unclear. This study examined the reproductive state of two Cerastoderma edule populations over a 6-month period to assess their reproductive condition, the incidence of intersex and presence of parasitic infection. A further seven native sites from south-west England were examined during the peak reproductive season to identify the presence of intersex within the region. Laboratory exposures of organisms collected from field populations showed a significantly female-biased sex ratio compared with controls when exposed to the endocrine disrupting chemicals, bisphenol-A (nominal concentration: 0.1 µg L−1) and 17β-oestradiol (nominal concentration: 0.1 µg L−1), but none of the chemical exposures induced intersex. Intersex was revealed in seven out of the nine native populations of C. edule sampled at peak reproductive season. The highest incidence and most severe case of intersex were reported at Lower Anderton on the River Tamer which also had a significantly female-biased sex ratio. Additionally, the dominant trematode family was the Bucephalaidae. Parasitic infection influences the maturity of C. edule by lowering both mean gonad index and condition index. These results suggest that endocrine disrupting chemicals could be contributing factors towards the development of intersex in C. edule.

Evaluation of the Chemical Durability of Iowa Fly Ash Concretes, HR-327, Phase II Progress Report, 1992

The major objective of this research project is to investigate how fly ash influences the chemical durability of portland cement based materials. The testing program is evaluating how Iowa fly ashes influence the sulfate durability of portland cement-fly ash pastes, mortars, and concretes. Also, alkali-reactivity studies are being conducted on mortar bar specimens prepared in accordance with ASTM C 311. Prelimary sulfate test results, based only on mortar bar studies, indicate that only the very high-calcium fly ash (29 percent CaO, by weight) consistently reduced the durability of test specimens exposed to a solution containing 5 percent sodium sulfate. The remaining four fly ashes that were used in the study showed negligible to dramatic increases in sulfate resistance. Concrete specimens were only beginning to respond to the sulfate solutions after about one year of exposure; and hence, considerably more time will be needed to assess their performance. Preliminary results from the alkali-reactivity tests have indicated that the Oreopolis aggregate is not sensitive to alkali attack. However, some of the test results have indicated that the testing procedure may be prone to delayed expansion due to the presence of periclase (MgO) in the Class C fly ashes. Research is being planned to: (1) verify if the periclase is influencing test results; and (2) estimating the magnitude of the potential error.

How to deal with uncertainties regarding the occupational exposure to antineoplastic mixtures: additive effect should always be considered?

Risk assessment considerations - The concept that “safe levels of exposure” for humans can be identified for individual chemicals is central to the risk assessment of compounds with known toxicological profiles. Selection of agents for combination chemotherapy regimens involves minimize overlapping of mechanisms of action, antitumor activity and toxicity profile. Although the toxicological profile and mechanism of action of each individual drug is well characterized, the toxicological interactions between drugs are likely, but poorly established at occupational exposure context. The synergistic nature of interactions may help in understanding the adverse health effects observed in healthcare workers, where exposure situations are characterized by complex mixtures of chemical agents, and the levels of individual exposing agents are often not sufficiently high to explain the health complaints. However, if a substance is a genotoxic carcinogen, this would be the “lead effect”; normally, no OEL based on a NOEL would be derived and the level would be set so low that it would be unlikely that other effects would be expected. Aim of the study - Recently research project developed in Portuguese Hospitals characterize the occupational exposure to antineoplastic agents and the health effects related. The project aimed to assess exposure of the different risk groups that handle antineoplastic agents in the hospital setting, namely during preparation and administration of these drugs. Here it is presented and discussed the results in a study developed in two hospitals from Lisbon.

Co-exposure to mycotoxins: the case of waste management workers

Introduction - Occupational exposures are characterized by being complex and associated to co-exposure to several contaminants by different exposure routes. Even if exposure occurs to only a chemical agent, it can have different exposure routes and can result in different health effects. The waste management setting is recognized by the presence of several chemical and biologic agents in the workplaces. Recently, it was reported occupational exposure to Aflatoxin B1 (AFB1) in one Portuguese waste management industry. However, data regarding to fungal burden showed that exposure to other mycotoxins should be expected. Aim of the study - The aim of the present work was to know if workers from this waste management industry were exposed to other mycotoxins besides AFB1.

Exposure Assessment for Formaldehyde and Acetaldehyde in the Workplace

Personal exposure and possible cancer risk to formaldehyde and acetaldehyde were appraised in 8 work places at a university in Brazil. Levels of formaldehyde measured ranged from 22.5 to 161.5 g·m 3 and from 18.3 to 91.2 g·m 3 for acetaldehyde. The personal exposure, expressed as the potential dose in indoor air, was calculated to range from 129.8 to 930.4 g·day 1 (low exposure) and 183.9 to 1318.1 g·day 1 (medium exposure) for formaldehyde and 105.5 to 525.3 g·day 1 (low exposure) and 149.5 to 744.2 g·day 1 (medium exposure) for acetaldehyde. The indoor/outdoor ratio showed the existence of indoor sources of the compounds which were mainly in practical classes and research laboratories. The highest formaldehyde and acetaldehyde levels were found where chemical reagents were manipulated. Relating the levels found to the permissible limit given by the US OSHA showed there was no particular risk although some formaldehyde levels did exceed the lower exposure limit of the US agency NIOSH. Any cancer risk would be highest for female technicians and teaching researchers.

Comet assay as a human biomonitoring tool: application in occupational exposure to antineoplastic drugs

Antineoplastic drugs are a heterogeneous group of chemicals used in the treatment of cancer, and have been proved by IARC to be mutagens, carcinogens and teratogens agents. In general, chemicals that interact directly with DNA by biding covalently or by intercalating, or indirectly by interfering with DNA synthesis, were among the first chemotherapeutics developed. Also, these drugs can induce reactive oxygen species that can lead to DNA damage and, consequently, mutations. These drugs are often used in combination to achieve synergistic effects on tumour cells resulting from their differing modes of action. However, most if not all of these chemical agents are generally nonselective and, along with tumour cells, normal cells may undergo cytotoxic/genotoxic damage. The in vivo exposure to antineoplastic drugs has been shown to induce different types of lesions in DNA, depending on the particular stage of cell cycle at the time of treatment. Besides the patients that use these drugs as a treatment, workers that handle and/or administer these drugs can be exposed to these substances; namely pharmacy, and nursing personnel in hospital context.

Application of mathematical models to mycotoxins children risk assessment: a case study of Portuguese children exposure to co-occurring mycotoxins in processed cereal-based foods

People, animals and the environment can be exposed to multiple chemicals at once from a variety of sources, but current risk assessment is usually carried out based on one chemical substance at a time. In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, namely mycotoxins, a wide group of fungal secondary metabolites that are known to potentially cause toxicity and carcinogenic outcomes. Mycotoxins are commonly found in a variety of foods including those intended for consumption by infants and young children and have been found in processed cereal-based foods available in the Portuguese market. The use of mathematical models, including probabilistic approaches using Monte Carlo simulations, constitutes a prominent issue in human health risk assessment in general and in mycotoxins exposure assessment in particular. The present study aims to characterize, for the first time, the risk associated with the exposure of Portuguese children to single and multiple mycotoxins present in processed cereal-based foods (CBF). Portuguese children (0-3 years old) food consumption data (n=103) were collected using a 3 days food diary. Contamination data concerned the quantification of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) were evaluated in 20 CBF samples marketed in 2014 and 2015 in Lisbon; samples were analyzed by HPLC-FLD, LC-MS/MS and GC-MS. Daily exposure of children to mycotoxins was performed using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) to the aflatoxin exposure. The magnitude of the MoE gives an indication of the risk level. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (ratio between exposure and a reference dose, HQ). For the cumulative risk assessment of multiple mycotoxins, the concentration addition (CA) concept was used. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. 71% of CBF analyzed samples were contaminated with mycotoxins (with values below the legal limits) and approximately 56% of the studied children consumed CBF at least once in these 3 days. Preliminary results showed that children exposure to single mycotoxins present in CBF were below the TDI. Aflatoxins MoE and MoET revealed a reduced potential risk by exposure through consumption of CBF (with values around 10000 or more). HQ and HI values for the remaining mycotoxins were below 1. Children are a particularly vulnerable population group to food contaminants and the present results point out an urgent need to establish legal limits and control strategies regarding the presence of multiple mycotoxins in children foods in order to protect their health. The development of packaging materials with antifungal properties is a possible solution to control the growth of moulds and consequently to reduce mycotoxin production, contributing to guarantee the quality and safety of foods intended for children consumption.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

Chemical characterization and in vitro cyto- and genotoxicity of ‘legal high’ products containing Kratom (Mitragyna speciosa)

Kratom is a popular ‘legal high’ mainly constituted by alkaloids extracted from the Mitragyna speciosa plant with mitragynine (MG) as the dominant active substance. The increasing use of Kratom for recreational purposes has alerted risk assessment bodies of the lack of information on the real composition and its potential health risks. The present study aimed to determine and compare the MG composition of 13 commercial products of Kratom sold online and in “smartshops”, by gas chromatography–mass spectrometry. For the first time, the cytotoxicity induced by pure MG and Kratom, extracts was evaluated in in vitro models of human intestinal (Caco-2) and neuronal (SH-SY5Y) cells after 6 and 24 h. Genotoxicity was also evaluated in intestinal Caco-2 cells following 24 h of exposure to subtoxic concentrations using the comet assay. The obtained results revealed an inconsistency between the information (‘power’) provided in labels and the MG content. Cytotoxicity tests revealed a concentration-dependent decrease in cell viability in both cellular models, with the SH-SY5Y cells being more sensitive to the Kratom extracts. The resin and the ‘powered extracts’ were the most cytotoxic samples, with IC50 values significantly lower than the leaf extracts and pure MG (P < 0.0001 vs. leaf extracts and MG). In addition, significant DNA damage was observed in Caco-2 cells exposed to these extracts but not to pure MG, which suggests that other substances present in the extracts or interactions involving Kratom components might be responsible for the observed effects.

An Assessment to Toxicological Risk of Pesticide Exposure

On the one hand, pesticides may be absorbed into the body orally, dermally, ocularly and by inhalation and the human exposure may be dietary, recreational and/or occupational where toxicity could be acute or chronic. On the other hand, the environmental fate and toxicity of the pesticide is contingent on the physico-chemical characteristics of pesticide, the soil composition and adsorption. Human toxicity is also dependent on the exposure time and individual’s susceptibility. Therefore, this work will focus on the development of an Artificial Intelligence based diagnosis support system to assess the pesticide toxicological risk to humanoid, built under a formal framework based on Logic Programming to knowledge representation and reasoning, complemented with an approach to computing grounded on Artificial Neural Networks. The proposed solution is unique in itself, once it caters for the explicit treatment of incomplete, unknown, or even self-contradictory information, either in terms of a qualitative or quantitative setting.