Statistical analysis of interactive cytotoxicity in human epidermal keratinocytes following exposure to a mixture of four metals

Exposure to mixtures of chemicals is an important and relevant environmental issue. Of particular interest is the detection and characterization of departure of biological effects from additivity. Methodology based on the assumption of additivity is used in fitting single-chemicaldata. Interactionsare determined and characterized by making comparisons between the observed and predicted responses at mixtures along a fixed ratio ray of the component substances. Two simultaneous tests are developed for testing for any departure from additivity. Multiple comparisons procedures are used to compare observed responses to that predicted under additivity. A simultaneous confidence band on the predicted responses along the mixture ray is also developed. The methods are illustrated with cytotoxicity data that arise when human epidermal keratinocytes are exposed to a mixture of arsenic, chromium, cadmium, and lead. Synergistic, antagonistic, and additive cytotoxicities were observed at different dose levels of the four-metal mixture.     

Disseminated Aspergillus flavus infection in broiler breeder pullets

Increased morbidity and mortality occurred in a 5-wk-old broiler breeder replacement pullet flock. The affected broiler pullet flock was housed on the first floor of a two-story confinement building. Mortality increased to 0.1%/day compared to the flock on the second floor, which had mortality levels of less than 0.01%/day. Clinical signs in the affected chickens included inactivity, decreased response to stimuli, and anorexia. No respiratory or neurologic signs were observed. On necropsy, affected pullets were dehydrated and emaciated and had disseminated variably sized single or multiple heterophilic granulomas that contained intralesional septate and branching fungal hyphae. Lesions were extensive around the base of the heart in the thoracic inlet and in the kidneys. Other affected organs included eyelid, muscle, proventriculus, ventriculus, intestine, liver, spleen, lung, and heart. Aspergillus flavus was cultured from the visceral granulomas. The source of flock exposure to the organism was not determined.     

Preservation causes shrinkage in seahorses: implications for biological studies and for managing sustainable trade with minimum size limits

• 1. The implications of shrinkage associated with desiccation and ethanol preservation for seahorses (genus Hippocampus) were investigated using Hippocampus guttulatus (European long‐snouted seahorse) as a model. Specifically, this research addressed the implications of preservation for taxonomy and life history studies and the application of minimum size limits (MSL) for managing seahorse trade.
• 2. In 2004, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) listed all seahorse species on its Appendix II, and recommended a 10 cm MSL as an interim means of ensuring sustainable international trade. Inconsistencies in seahorse measurement methods and repeatability posed challenges for applying the MSL. Moreover, the shrinking effect of desiccation on body length observed in other fish was assumed to be negligible for seahorses because of their high degree of ossification.
• 3. Changes in seahorse sizes were measured following immersion in ethanol and desiccation. H. guttulatus shrank on average by 0.1–2.3% when preserved in ethanol and 3.0–6.4% when dried, depending on the trait measured. Similar trends were observed in a sample of H. kuda (yellow seahorse). Specimen posture during drying, and measurement methods also influenced estimates of size.
• 4. Based on the shrinkage observed, 14–44% of captured seahorses that are dried could shrink to below the recommended MSL, even if all seahorses were longer than the MSL at capture. This demonstrates that small changes in body lengths can have significant implications for trade of species managed with size limits.
• 5. Recommendations are to (1) standardize seahorse measurement methods, (2) consider the effects of preservation and measurement technique on body lengths, and apply appropriate corrections in comparative studies and when developing fisheries management strategies, and (3) adjust size limits at the point of capture to ensure retained seahorses comply with the CITES recommended MSL.

Copyright © 2008 John Wiley & Sons, Ltd.     

A quantitative assessment of the BSE risk associated with fly ash and slag from the incineration of meat-and-bone meal in a gas-fired power plant in Denmark

It has been recommended that meat-and-bone meal (MBM) be incinerated at 850 °C for at least 2 s and the ashes and slag disposed of in controlled landfills, to dispose of animal-derived proteins. Most commonly, the MBM is incinerated in cement works or coal-fired power plants and the ashes and slag are incorporated into the cement or concrete.

Our goal was to assess with a Monte Carlo simulation model the bovine spongiform encephalopathy (BSE) risk to cattle and humans posed by the ash and slag. The results will be used by decision makers to evaluate the need for disposal of the fly ash in controlled landfills and the feasibility of use of the ash by the phosphate and fertilizer industries.

We assumed that all specified risk material (SRM) and MBM produced in Denmark would be incinerated in this gas-fired power plant. Based on observations in 2001, we assumed that, on average, six (range: 0–15) clinical BSE cases each year were rendered into MBM and incinerated. In addition, SRM or carcasses from 0 to 31 (median = 10) BSE-infected-but-undetected animals/BSE case were also incinerated.

The simulations were run on a 1-week basis. Our results suggest that if the slag is collected and re-incinerated the median BSE infectivity remaining in the fly ash per week would be 3.1E−11 cattle ID₅₀. A cattle ID₅₀ is the amount of infectivity that will cause infection in 50% of cattle exposed to it. During the weeks when BSE was infected in the SRM-MBM, the median infectivity in the fly ash was estimated as 8.7E−10 cattle ID₅₀ and 2.9E−12 human ID₅₀. The 95th percentiles were 2.1E−08 cattle ID₅₀ and 5.8E−10 human ID₅₀, respectively. One ton of fly ash would contain ≤1.8E−07 cattle ID₅₀ 95% of the time. These are the potential exposures of the cattle or human populations. The potential exposures of individuals are far less.     

Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants

OBJECTIVE: To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing. SAMPLE POPULATION: 2 ejaculates collected from each of 5 stallions. PROCEDURE: Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-tocopherol (0.5% ethanol). After thawing, spermatozoal motility was assessed via computer-assisted analysis and DNA fragmentation was assessed via the comet assay. Spermatozoal mitochondrial membrane potential, acrosomal integrity, and viability were determined by use of various specific staining techniques and flow cytometry. RESULTS: The addition of enzyme scavengers or antioxidants to cryopreservation extender did not improve spermatozoal motility, DNA fragmentation, acrosomal integrity, viability, or mitochondrial membrane potential after thawing. Superoxide dismutase increased DNA fragmentation, likely because of the additional oxidative stress caused by the generation of hydrogen peroxide by this enzyme. Interestingly, the addition of the vehicle for alpha-tocopherol resulted in a significant decrease in live acrosome-intact spermatozoa. CONCLUSIONS AND CLINICAL RELEVANCE: The addition of antioxidants to the cryopreservation extender did not improve the quality of equine spermatozoa after thawing, which suggests that the role of oxidative stress in cryopreservation-induced damage of equine spermatozoa requires further investigation. Our data suggest that solubilizing alpha-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of alpha-tocopherol may be preferred for future investigations.     

The development of methods for immunophenotypic and lymphocyte function analyzes for assessment of Southern sea otter (Enhydra lutris nereis) health

The Southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act. The population began a pattern of slow decline in 1995. The decline was attributed to high adult mortality rates with infectious disease being the major cause of death. Multiple pathogens were implicated in these deaths including opportunistic pathogens such as Coccidiodes immitis and Toxoplasma sp. These findings suggested that the immunological health of mature animals in this population might be compromised. The primary goal of this study was to establish techniques for assessing phenotypic and functional baseline data for peripheral blood mononuclear cells (PBMC) in free-ranging sea otters. Standard total and differential white blood cell counts were augmented by emumeration of T and B lymphocyte subsets. Lymphocyte function was determined by both mitogen-induced proliferation and expression of IL-2 receptors. In addition to establishing normal ranges for adult animals, age-related changes were identified in B lymphocyte numbers and cell-surface density of major histocompatability complex class II (MHC II) proteins. The predominant lymphocyte subpopulation in Southern sea otters is the T lymphocyte. Substantial variation among individual animals was observed within the B lymphocyte population both in cell number and density of MHC II expression. Pups had greater numbers of T and B lymphocyte, as well as, greater MHC II expression on B lymphocytes than adults. Mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC) was variable among individual animals with no significant difference in cell response between age class and gender. Concanavalin (ConA) was a more effective mitogen in stimulating proliferation and interleukin (IL)-2 receptor expression than pokeweed. This data can be used to augment routine hematology profiles and aid in the identification of animals with immunologic perturbations.