Assessing the exposure risk and impacts of pharmaceuticals in the environment on individuals and ecosystems

The use of human and veterinary pharmaceuticals is increasing. Over the past decade, there has been a proliferation of research into potential environmental impacts of pharmaceuticals in the environment. A Royal Society-supported seminar brought together experts from diverse scientific fields to discuss the risks posed by pharmaceuticals to wildlife. Recent analytical advances have revealed that pharmaceuticals are entering habitats via water, sewage, manure and animal carcases, and dispersing through food chains. Pharmaceuticals are designed to alter physiology at low doses and so can be particularly potent contaminants. The near extinction of Asian vultures following exposure to diclofenac is the key example where exposure to a pharmaceutical caused a population-level impact on non-target wildlife. However, more subtle changes to behaviour and physiology are rarely studied and poorly understood. Grand challenges for the future include developing more realistic exposure assessments for wildlife, assessing the impacts of mixtures of pharmaceuticals in combination with other environmental stressors and estimating the risks from pharmaceutical manufacturing and usage in developing countries. We concluded that an integration of diverse approaches is required to predict ‘unexpected’ risks; specifically, ecologically relevant, often long-term and non-lethal, consequences of pharmaceuticals in the environment for wildlife and ecosystems.     

Aging and death in an organism that reproduces by morphologically symmetric division

In macroscopic organisms, aging is often obvious; in single-celled organisms, where there is the greatest potential to identify the molecular mechanisms involved, identifying and quantifying aging is harder. The primary results in this area have come from organisms that share the traits of a visibly asymmetric division and an identifiable juvenile phase. As reproductive aging must require a differential distribution of aged and young components between parent and offspring, it has been postulated that organisms without these traits do not age, thus exhibiting functional immortality. Through automated time-lapse microscopy, we followed repeated cycles of reproduction by individual cells of the model organism Escherichia coli, which reproduces without a juvenile phase and with an apparently symmetric division. We show that the cell that inherits the old pole exhibits a diminished growth rate, decreased offspring production, and an increased incidence of death. We conclude that the two supposedly identical cells produced during cell division are functionally asymmetric; the old pole cell should be considered an aging parent repeatedly producing rejuvenated offspring. These results suggest that no life strategy is immune to the effects of aging, and therefore immortality may be either too costly or mechanistically impossible in natural organisms.     

Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium

Exposure of cultured bovine pulmonary artery endothelial cells to varying levels of hypoxia (10% or 0% O2) for 4 hours resulted in a significant dose-dependent inhibition in endothelial prostacyclin synthesis (51% and 98%, at the 10% and 0% O2 levels respectively, p less than 0.05, compared to 21% O2 exposure values). Release of 3H-arachidonic acid from cellular pools was not altered by hypoxia. Some of the cells were incubated with arachidonic acid (20 microM for 5 min) or PGH2 (4 microM for 2 min) immediately after exposure. Endothelium exposed to 0% O2, but not to 10% O2, produced significantly less prostacyclin after addition of either arachidonic acid (25 +/- 5% of 21% O2 exposure values, n = 6, p less than 0.01) or PGH2 (31 +/- 3% of 21% O2 exposure values, n = 6, p less than 0.05). These results suggest that hypoxia inhibits cyclooxygenase at the 10% O2 level and both cyclooxygenase and prostacyclin synthetase enzymes at the 0% O2 exposure levels. Exposure of aortic endothelial cells resulted in a 44% inhibition of prostacyclin at the 0% exposure level. No significant alteration in prostacyclin production was found in pulmonary vascular smooth muscle cells exposed to hypoxia. These data suggest that the increased prostacyclin production reported in lungs exposed to hypoxia is not due to a direct effect of hypoxia on the main prostacyclin producing cells of the pulmonary circulation.     

Cyclooxygenase contracting factors and altered pulmonary vascular responses in chronically hypoxic newborn pigs.

Pulmonary hypertension and blunted pulmonary vascular responses to ACh develop when newborn pigs are exposed to chronic hypoxia for 3 days. To determine whether a cyclooxygenase (COX)-dependent contracting factor, such as thromboxane, is involved with altered pulmonary vascular responses to ACh, newborn piglets were raised in 11% O(2) (hypoxic) or room air (control) for 3 days. Small pulmonary arteries (100-400 microm diameter) were cannulated and pressurized, and their responses to ACh were measured before and after either the COX inhibitor indomethacin; a thromboxane synthesis inhibitor, dazoxiben or feregrelate; or the thromboxane-PGH(2)-receptor antagonist SQ-29548. In control arteries, indomethacin reversed ACh responses from dilation to constriction. In contrast, hypoxic arteries constricted to ACh before indomethacin and dilated to ACh after indomethacin. Furthermore, ACh constriction in hypoxic arteries was nearly abolished by either dazoxiben, feregrelate, or SQ-29548. These findings suggest that thromboxane is the COX-dependent contracting factor that underlies the constrictor response to ACh that develops in small pulmonary arteries of piglets exposed to 3 days of hypoxia. The early development of thromboxane-mediated constriction may contribute to the pathogenesis of chronic hypoxia-induced pulmonary hypertension in newborns.     

Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process

The Quality by Design (QbD) approach to the production of therapeutic monoclonal antibodies (mAbs) emphasizes an understanding of the production process ensuring product quality is maintained throughout. Current methods for measuring critical quality attributes (CQAs) such as glycation and glycosylation are time and resource intensive, often, only tested offline once per batch process. Process analytical technology (PAT) tools such as Raman spectroscopy combined with chemometric modeling can provide real time measurements process variables and are aligned with the QbD approach. This study utilizes these tools to build partial least squares (PLS) regression models to provide real time monitoring of glycation and glycosylation profiles. In total, seven cell line specific chemometric PLS models; % mono-glycated, % non-glycated, % G0F-GlcNac, % G0, % G0F, % G1F, and % G2F were considered. PLS models were initially developed using small scale data to verify the capability of Raman to measure these CQAs effectively. Accurate PLS model predictions were observed at small scale (5 L). At manufacturing scale (2000 L) some glycosylation models showed higher error, indicating that scale may be a key consideration in glycosylation profile PLS model development. Model robustness was then considered by supplementing models with a single batch of manufacturing scale data. This data addition had a significant impact on the predictive capability of each model, with an improvement of 77.5% in the case of the G2F. The finalized models show the capability of Raman as a PAT tool to deliver real time monitoring of glycation and glycosylation profiles at manufacturing scale.     

Impedance as an alternative to MPN enumeration of coliforms in pasteurized milks

R.H. MADDEN AND A. GILMOUR. 1995. Samples (900) of pasteurized whole, semi-skimmed and skimmed milk were subjected to conventional enumeration of coliforms by a nine-tube most probable number (MPN) technique, and impedance enumeration, in parallel. Regression analysis of the positive samples (98) showed that impedance enumeration was at least as accurate as the MPN method but results were obtained faster, with all testing being completed in 20 h, rather than 48 h. Consumable requirements, and staffing levels, were also much less with the impedance system. The impedance method could therefore beneficially replace the conventional method.     

Foodborne enterotoxigenic Escherichia coli: detection and enumeration by DNA colony hybridization

Four methods were compared for detecting heat-labile toxin production by Escherichia coli: DNA colony hybridization, two enzyme-linked immunosorbent assays, and the mouse Y-1 adrenal cell reaction. Although results of the methods were in general agreement, there were some differences in specificity and sensitivity. DNA colony hybridization was used to detect and enumerate enterotoxigenic E. coli isolates in artificially contaminated food without enrichment. Sensitivity level was 100 cells per g.     

Resistance and susceptibility of Boronia megastigma cultivars to infestations by the psyllid Ctenarytaina thysanura

Studies were conducted on 23 boronia cultivars to determine the mechanisms of resistance and susceptibility to the psyllid, Ctenarytaina thysanura Ferris and Klyver. No antibiosis effects were detected in any of the boronia cultivars investigated. No cultivar was tolerant to C. thysanura attack. However, in both free-choice and no-choice tests in the glasshouse, ovipositional antixenosis (non-preference) was detected in cultivar HC27, whereas cultivars HC4 and HC142 were most preferred for oviposition. Measurement of terminal shoot hardness of cultivars revealed variations in relative hardness. Given a choice between boronia cultivars of the same size and colour but of differing hardness of the terminal shoots, C. thysanura laid more eggs on those cultivars with softer terminal shoots (HC4 and HC142) and laid fewest eggs on harder terminal shoots (HC27). This may explain differences in the psyllid population on different cultivars in the field. Fewer eggs were laid on terminal shoots exceeding a hardness rating of 80 g/mm terminal shoot thickness.