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USGS2010

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers.

Zygote↗

USGS2018

A comparison of the chemical sensitivities between in vitro and in vivo propagated juvenile freshwater mussels: Implications for standard toxicity testing

Unionid mussels are ecologically important and are globally imperiled. Toxicants contribute to mussel declines, and toxicity tests using juvenile mussels—a sensitive life stage—are valuable in determining thresholds used to set water quality criteria. In vitro culture methods provide an efficient way to propagate juveniles for toxicity testing, but their relative chemical sensitivity compared with in vivo propagated juveniles is unknown. Current testing guidelines caution against using in vitro cultured juveniles until this sensitivity is described. Our objective was to evaluate the relative sensitivity of juvenile mussels produced from both in vitro and in vivo propagation methods to selected chemicals. We conducted 96-h acute toxicity tests according to ASTM International guidelines with 3 mussel species and 6 toxicants: chloride, nickel, ammonia, and 3 copper-based compounds. Statistically significant differences between in vitro and in vivo juvenile 96-h median effect concentrations were observed in 8 of 17 tests, and in vitro juveniles were more sensitive in 6 of the 8 significant differences. At 96 h, 4 of the 8 statistically different tests for a given chemical were within a factor of 2, which is the intralaboratory variation demonstrated in a recent evaluation of mussel toxicity tests. We found that although differences in chemical sensitivity exist between in vitro and in vivo propagated juvenile mussels, they are within normal toxicity test variation. Therefore, in vitro propagated juvenile mussels may be appropriate for use in ASTM International-based toxicity testing.

Environmental Toxicology and Chemistry↗

USGS2017

Occurrence and in vitro bioactivity of estrogen, androgen, and glucocorticoid compounds in a nationwide screen of United States stream waters

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a broad suite of chemical analytes, in streamwater from 35 well-characterized sites (3 reference and 32 impacted) across 24 states and Puerto Rico. ER agonism was the most frequently detected with nearly all sites (34/35) displaying activity (range, 0.054–116 ng E2Eq L –1 ). There was a strong linear relationship ( r 2 = 0.917) between in vitro ER activity and concentrations of steroidal estrogens after correcting for the in vitro potency of each compound. AR agonism was detected in 5/35 samples (range, 1.6–4.8 ng DHTEq L –1 ) but concentrations of androgenic compounds were largely unable to account for the in vitro activity. Similarly, GR agonism was detected in 9/35 samples (range, 6.0–43 ng DexEq L –1 ); however, none of the recognized GR-active compounds on the target-chemical analyte list were detected. The utility of in vitro assays in water quality monitoring was evident from both the quantitative agreement between ER activity and estrogen concentrations, as well as the detection of AR and GR activity for which there were limited or no corresponding target-chemical detections to explain the bioactivity. Incorporation of in vitro bioassays as complements to chemical analyses in standard water quality monitoring efforts would allow for more complete assessment of the chemical mixtures present in many surface waters.

Environmental Science & Technology↗

USGS2016

VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro . These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

Infection, Genetics and Evolution↗

USGS2010

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines

Theriogenology↗

USGS1991

Responses to in vitro and in vivo immunisations with Aeromonas salmonicida O antigen bacterins in rainbow trout (Oncorhynchus mykiss)

Immunological assays were developed to test for the effects of immunisation with Aeromonas salmonicida O antigen bacterin in rainbow trout ( Oncorhynchus mykiss ) in vitro and in vivo . Trout spleen sections were immunised in vitro by incubation in media containing 100, 10, and 1 μ g ml −1 of the A. salmonicida O antigen. After 10 days at 15°C, the individual sections were minced and cell suspensions assayed for the effects of the bacterin on non-specific and specific immune responses. Assays of the cell suspensions showed that the neutrophil oxidative activity, phagocytic capabilities, numbers of plaque-forming cells (PFC) and circulating antibody titres all increased in proportion to the bacterin dosage. Trout immunised in vivo with 100 μ g by injection and by 100 μ g ml −1 bath with the O antigen, showed similar responses to spleen sections immunised in vitro . The appearance of non-specific PFC against unlabelled sheep red blood cells was highest in spleens immunised in vitro and in the fish immunised by injection; non-specific PFC rarely occurred in O antigen bathed fish and in control fish. The development of these assays may enable biologists to follow the effects on the non-specific defence mechanisms and the specific immune response in cultivated fish populations after, immunisation with A. salmonicida bacterins and thus aid in the development of protective vaccination against furunculosis.

Fish and Shellfish Immunology↗

USGS1991

In vivo and in vitro transfer of trout spleen sections for early analysis of the immune response

To determine the earliest time after in vivo immunisation that the spleen could be excised and held in vitro to detect an immune response, lake trout ( Salvelinus namaycush ) were exposed to DNP-Ficoll or Yersinia ruckeri O antigen administered by intraperitoneal injection or by bath. The spleens were excised from the fish at selected times after immunisation, placed in vitro and held in tissue culture media at 14°C until 10 days after the in vivo immunisation. They then were analysed for an immune response by counting the numbers of plaqueforming cells (PFC). PFC were first detected in samples taken 3 days after injection with either antigen. With bath immunisation, however, PFC were first seen 6 days post-immunisation when using the Y. ruckeri O antigen and no PFC above background levels appeared in fish bathed in the DNP-Ficoll solution. On day 10 after immunisation, the spleens taken directly from immunised fish always produced more PFC than when the spleens were taken through in vitro culture. A unique feature of this method, is that an immune response to an antigen may be detected by excising the spleen and holding it in in vitro culture without necessarily holding the fish for long periods of time after antigen injection or bath. This technique also adds more information on how rapidly the bacterins or antigens are processed by fish to initiate an immune response.

Fish and Shellfish Pathology↗

USGS2009

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

Hawai'i↗

USGS2014

Integrated assessment of runoff from livestock farming operations: analytical chemistry, in vitro bioassays, and in vivo fish exposures

Animal waste from livestock farming operations can contain varying levels of natural and synthetic androgens and/or estrogens, which can contaminate surrounding waterways. In the present study, surface stream water was collected from 6 basins containing livestock farming operations. Aqueous concentrations of 12 hormones were determined via chemical analyses. Relative androgenic and estrogenic activity was measured using in vitro cell assays (MDA-kb2 and T47D-Kbluc assays, respectively). In parallel, 48-h static-renewal in vivo exposures were conducted to examine potential endocrine-disrupting effects in fathead minnows. Mature fish were exposed to surface water dilutions (0%, 25%, 50%, and 100%) and 10-ng/L of 17α-ethynylestradiol or 50-ng/L of 17β-trenbolone as positive controls. Hepatic expression of vitellogenin and estrogen receptor α mRNA, gonadal ex vivo testosterone and 17β-estradiol production, and plasma vitellogenin concentrations were examined. Potentially estrogenic and androgenic steroids were detected at low nanogram per liter concentrations. In vitro estrogenic activity was detected in all samples, whereas androgenic activity was detected in only 1 sample. In vivo exposures to the surface water had no significant dose-dependent effect on any of the biological endpoints, with the exception of increased male testosterone production in 1 exposure. The present study, which combines analytical chemistry measurements, in vitro bioassays, and in vivo fish exposures, highlights the integrated value and future use of a combination of techniques to obtain a comprehensive characterization of an environmental chemical mixture.

Arkansas, Iowa, Kentucky, New York, Wisconsin↗

USGS1996

In vivo/in vitro comparison of pharmacokinetics and pharmacodynamics of 3,3',4,4'-tetrachlorobiphenyl (PCB77)

The rat hepatoma cell line, H4IIE, serves as a useful tool to assess potential biological effects such as induction of cytochrome P4501A1 expression. The objectives of this study were twofold: to investigate the kinetic time course and dosimetry of PCB77 in rat hepatoma cells dosed with PCB77 and in liver of rats given ip doses of PCB77, and to compare in vitro and in vivo P4501A1 enzyme induction responses. For the 4-day time–course study, H4IIE cells were exposed with two doses of [ 14 C]PCB77 (0.9 and 3 μg/plate) and harvested at 15 and 30 min, 1, 2, 4, 8, and 12 hr, and 1, 2, 3, and 4 days. PCB77-derived radioactivity was detected in the cells as early as 15 min postdosing. For the dose–response study, the cells were dosed with various concentrations of PCB77 (0.00316–5.37 μg/plate) and harvested on Day 3 since ethoxyresorufin O -deethylase (EROD) activity in vitro reached its maximum on the third day postdosing. Time–course and dose–response studies revealed that only 1–3% of the total delivered dose was found in the cells, with the remainder in the media and adhering to the culture plates. For the dose–response study in vivo, male Fischer rats were dosed with a single ip injection of various concentrations of PCB77 (0.1–50 mg/kg body wt) and euthanized on Day 3. PCB77-derived radioactivity and EROD induction in vivo were measured. When EROD activity and PCB77-derived radioactivity in the rat hepatoma cells and in the rat liver were compared on an equivalent weight basis, there was a significant correlation ( r 2 = 0.985) between them. Prior to this study, no information on quantitative dosimetry and EROD activities of PCB77 has been reported to validate the in vitro assay with in vivo data.

Toxicology and Applied Pharmacology↗

USGS2017

Comparison of in vitro estrogenic activity and estrogen concentrations insource and treated waters from 25 U.S. drinking water treatment plants

In vitro bioassays have been successfully used to screen for estrogenic activity in wastewater and surface water, however, few have been applied to treated drinking water. Here, extracts of source and treated water samples were assayed for estrogenic activity using T47D-KBluc cells and analyzed by liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) for natural and synthetic estrogens (including estrone, 17β-estradiol, estriol, and ethinyl estradiol). None of the estrogenswere detected above the LC-FTMS quantification limits in treated samples and only 5 source waters had quantifiable concentrations of estrone, whereas 3 treated samples and 16 source samples displayed in vitro estrogenicity. Estrone accounted for themajority of estrogenic activity in respective samples, however the remaining samples that displayed estrogenic activity had no quantitative detections of known estrogenic compounds by chemical analyses. Source water estrogenicity (max, 0.47 ng 17β-estradiol equivalents (E2Eq) L−1) was below levels that have been linked to adverse effects in fish and other aquatic organisms. Treated water estrogenicity (max, 0.078 ng E2Eq L−1) was considerably below levels that are expected to be biologically relevant to human consumers. Overall, the advantage of using in vitro techniques in addition to analytical chemical determinations was displayed by the sensitivity of the T47D-KBluc bioassay, coupled with the ability tomeasure cumulative effects of mixtures, specifically when unknown chemicals may be present.

Science of the Total Environment↗

USGS2023

Establishing an in vitro model to assess the toxicity of 6PPD-quinone and other tire wear transformation products

The tire wear transformation product 6PPD-quinone (6PPDQ) has been implicated as the causative factor for broad scale mortality events for coho salmon in the Pacific Northwest. Highly variable sensitivity to 6PPDQ in closely related salmonids complicates efforts to evaluate the broader toxicological impacts to aquatic ecosystems. Our goals were to (1) validate the large range of in vivo species sensitivities reported for coho, Chinook, and sockeye salmon and (2) develop an in vitro platform for assessing 6PPDQ toxicity. In vivo studies confirmed the acute sensitivity of juvenile coho (12 h LC 50 = 80.4 ng/L) and demonstrated that sockeye salmon were not vulnerable to mortality. Chinook salmon were sensitive to 6PPDQ mortality at initial concentrations >25 μg/L, ∼10-fold greater than reported environmental measurements. In vitro , the coho salmon cell line CSE-119 was acutely sensitive to 6PPDQ (metabolic EC 50 = 7.9 μg/L, cytotoxicity EC 50 = 6.1 μg/L). Analogous Chinook (CHSE-214) and sockeye salmon (SSE-5) cell lines were nonresponsive in both assays, and rainbow trout RTG-2 cells began showing metabolic effects at 68 μg/L (EC 5 ). Recreation of species-specific 6PPDQ sensitivity in vitro implicates conserved modes of action in CSE-119 that could be utilized for mechanistic studies of 6PPDQ toxicity and screening of other PPD transformation products.

Environmental Science and Technology Letters↗

USGS1974

In vitro and in vivo efficacy of potentiated sulfonamide in control of furunculosis in salmonids

In vitro and in vivo efficacy of a potentiated sulffonamide, Ro5–0037, for inhibition of Aeromonas salmonicida , causative agent of fish furunculosis was determined. The components of the potentiated sulfonamide, sulfadimethoxine, and ormetoprim were tested alone and in combination. In vitro results showed that both sulfonamide-resistant and sensitive strains of A . salmonicida had greater sensitivity to Ro5–0037 than to either of its components. Also, sulfonamide-sensitive strains developed resistance to ormetoprim and sulfadimethoxine more rapidly than to Ro5–0037. Efficacy studies with small numbers of artificially infected trout revealed that Ro5–0037 effectively controlled furunculosis at 50 mg/kg of fish/day, one fourth the usual sulfonamide dosage level. Extensive field trials also showed the 50 mg/kg level of Ro5–0037 gave effective control of hatchery outbreaks of furunculosis. As found in the in vitro studies, all in vivo trials indicated the combination of ormetoprim and sulfadimethoxine was more efficient in inhibiting A . salmonicida than either component alone.

Journal of the Fisheries Research Board of Canada↗

USGS2002

PCR und ELISA - Alternativen zum Maustest für die Analyse des Botulismus-Neurotoxin-C1 Giftbildungspotentiales in Umweltproben? [PCR and ELISA - in vitro alternatives to the mouse-bioassay for assessing the botulinum-neurotoxin-C1 production potential in environmental samples?]

Botulism is one of the most important bird diseases world-wide and is caused by the intoxication with Botulinum-Neurotoxin-C1 (BoNt-C1), which is produced by toxigenic clostridia under appropriate conditions. Avian botulism leads regularly to large losses among the migrating bird populations breeding and resting at the saltwater pools of the Austrian national park Neusiedler See-Seewinkel. Despite of its ethical dubiousness and its high technical expense the mouse-bioassay is still used as the routine standard method for the detection of BoNt-C1. According to the 3R-concept, in vitro alternative methods for the qualitative detection of BoNt-C1 (immunostick-ELISA) and a corresponding BoNt-C1 gene fragment (nested-PCR) were established. In order to estimate the BoNt-C1 production potential the methods were tested with sediment samples from different saltwater pools subjected to cultivation conditions appropriate for in vitro BoNt-C1-production. With the mouse-bioassay, 52 out of 77 samples were found to have a positive toxin production potential. The immunostick-ELISA showed a similar sensitivity as the mouse-bioassay and exhibited a highly significant positive correlation (r=0.94; p<0.001) with the mouse-bioassay in detecting BoNt-C1. The nested-PCR approach revealed higher numbers of positive BoNt-C1 gene fragment detections as compared to the direct toxin analysis approaches. A weak correlation (r=0.21; p=0.07) with the mouse-bioassay was discernible, no correlation was found with the immunostick-ELISA (r=0.09; p=0.46). Obviously, the PCR approach detected the BoNt-C1 gene fragment in some of the samples where no toxin expression has occurred. Thus it is suggested that the qualitative immunostick-ELISA represents a potential in vitro alternative to the mouse-bioassay for assessing the BoNt-C1 production potential in environmental samples. In contrast, qualitative BoNt-C1 gene fragment detection via PCR led to an overestimation of the actual toxin production potential.

Neusiedler See-Seewinkel National Park↗

USGS1979

Allantoinase in lake trout ( Salvelinus namaycush ): In vitro effects of PCBs, DDT and metals

1. Allantoinase, an enzyme in the purine-urea cycle, was found in livers of Salvelinus namaycush (Osteichthyes: Salmoniformes). 2. The enzyme was active from pH 6.6 to 8.2 at 37°C and from pH 7.4 to 9.0 at 10°C and had an Arrhenius energy was activation of 11.0 kcal/mol and a temperature quotient of 2.0. The K m of the enzyme homogenate was 8.4 mM allantoin. 3. The concentrations of inorganic metals at which 50% inhibition occurred during in vitro exposure were 6.0 mg/l Cu 2+ , 6.7 mg/l Cd 2+ , 34 mg/l Hg 2+ and 52 mg/l Pb 2+ . The in vitro sensitivity to PCBs, DDT and DDE and kinetics in the presence of metals were determined. 4. Allantoinase activity was negatively correlated with body length for fish from Lake Michigan but not from Lake Superior or the laboratory.

Comparative Biochemistry and Physiology, Part C: C↗

USGS2025

Establishment of a cell culture from Daphnia magna as an in vitro model for (eco)toxicology assays: Case study using Bisphenol A as a representative cytotoxic and endocrine disrupting chemical

Bisphenol A (BPA) is a widely used industrial compound found in polycarbonate plastics, epoxy resin, and various polymer materials, leading to its ubiquitous presence in the environment. The toxicity of BPA to aquatic organisms has been well documented following in vivo exposure scenarios, with known cytotoxic and endocrine-disrupting effects. As such, BPA was used in this study as a well-characterized chemical to implement more ethical and resource-efficient scientific practices in toxicity testing through new approach methods (NAMs). Due to the frequent use of Daphnia spp. as a model organism in toxicology, we developed an in vitro cell culture system from Daphnia magna embryos, with optimized medium to support cell longevity. The cultures were maintained for up to two months, demonstrating their stability and suitability for cytotoxicity studies. Using this novel system, lethal concentration 50 (LC 50 ) values were determined at the 24 and 48 h time points following BPA exposure. Subsequently, oxidative stress, endocrine disruption, and DNA damage were assessed through gene expression, activity assays, and a comet assay in BPA-exposed cells. LC 50 values of 52 µM and 20 µM BPA were calculated after 24 and 48 h exposures, respectively. BPA cells exposed to 20 and 52 µM had significantly increased GSH, GPx, and GST activity levels. mRNA expression analysis revealed significant upregulations in the expression of hsp70, hsp90, gst, gpx, vtg1 , and cyp4 , with downregulations of sod, cat , and ecr following BPA exposure. Furthermore, comet assays showed a significantly higher level of DNA damage induced by BPA compared to controls, with greater comet and tail lengths. This study established a novel in vitro Daphnia model, using BPA as a case study for determining toxic effects, further highlighting the importance and applicability of utilizing alternative methods in ecotoxicological research through reducing animal use.

Aquatic Toxicology↗

USGS2017

The effect of aluminium and sodium impurities on the in vitro toxicity and pro-inflammatory potential of cristobalite

Background Exposure to crystalline silica (SiO 2 ), in the form of quartz, tridymite or cristobalite, can cause respiratory diseases, such as silicosis. However, the observed toxicity and pathogenicity of crystalline silica is highly variable. This has been attributed to a number of inherent and external factors, including the presence of impurities. In cristobalite-rich dusts, substitutions of aluminium (Al) for silicon (Si) in the cristobalite structure, and impurities occluding the silica surface, have been hypothesised to decrease its toxicity. This hypothesis is tested here through the characterisation and in vitro toxicological study of synthesised cristobalite with incremental amounts of Al and sodium (Na) dopants. Methods Samples of synthetic cristobalite with incremental amounts of Al and Na impurities, and tridymite, were produced through heating of a silica sol-gel. Samples were characterised for mineralogy, cristobalite purity and abundance, particle size, surface area and surface charge. In vitro assays assessed the ability of the samples to induce cytotoxicity and TNF-α production in J774 macrophages, and haemolysis of red blood cells. Results Al-only doped or Al+Na co-doped cristobalite contained between 1 and 4 oxide wt% Al and Na within its structure. Co-doped samples also contained Al- and Na-rich phases, such as albite. Doping reduced cytotoxicity to J774 macrophages and haemolytic capacity compared to non-doped samples. Al-only doping was more effective at decreasing cristobalite reactivity than Al+Na co-doping. The reduction in the reactivity of cristobalite is attributed to both structural impurities and a lower abundance of crystalline silica in doped samples. Neither non-doped nor doped crystalline silica induced production of the pro-inflammatory cytokine TNF-α in J774 macrophages. Conclusions Impurities can reduce the toxic potential of cristobalite and may help explain the low reactivity of some cristobalite-rich dusts. Whilst further work is required to determine if these effects translate to altered pathogenesis, the results have potential implications for the regulation of crystalline silica exposures.

Environmental Research↗

USGS2014

In vivo and in vitro phenotypic differences between Great Lakes VHSV genotype IVb isolates with sequence types vcG001 and vcG002

Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus first recognized in farmed rainbow trout in Denmark. In the past decade, a new genotype of this virus, IVb was discovered in the Laurentian Great Lakes basin and has caused several massive die-offs in some of the 28 species of susceptible North American freshwater fishes. Since its colonization of the Great Lakes, several closely related sequence types within genotype IVb have been reported, the two most common of which are vcG001 and vcG002. These sequence types have different spatial distributions in the Great Lakes. The aim of this study was to determine whether the genotypic differences between representative vcG001 (isolate MI03) and vcG002 (isolate 2010-030 #91) isolates correspond to phenotypic differences in terms of virulence using both in vitro and in vivo approaches. In vitro infection of epithelioma papulosum cyprini (EPC), bluegill fry (BF-2), and Chinook salmon embryo (CHSE) cells demonstrated some differences in onset and rate of growth in EPC and BF-2 cells, without any difference in the quantity of RNA produced. In vivo infection of round gobies ( Neogobius melanostomus ) via immersion exposure to different concentrations of vcG001 or vcG002 caused a significantly greater mortality in round gobies exposed to 10 2 plaque forming units ml − 1 of vcG001. These experiments suggest that there are phenotypic differences between Great Lakes isolates of VHSV genotype IVb.

Journal of Great Lakes Research↗