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At least 37 records · Page 2Linked to original sources

USGS1999

Identification of in vitro cytochrome P450 modulators to detect induction by prototype inducers in the mallard duckling ( Anas platyrhynchos

Seven modulators of mammalian monooxygenase activity were screened for their ability to selectively stimulate or inhibit in vitro monooxygenase activities of hepatic microsomes from mallard ducklings treated with phenobarbital, β -naphthoflavone, 3,3′,4,4′,5-pentachlorobiphenyl or vehicle. Microsomes were assayed fluorometrically for four monooxygenases: benzyloxy-, ethoxy-, methoxy-, and pentoxyresorufin- O -dealkylase, in combination with each of the seven modulators. Four combinations: α -naphthoflavone and 2-methylbenzimidazole with benzyloxyresorufin, and Proadifen with methoxy- and ethoxyresorufin, respectively, were evaluated further. β -Naphthoflavone-treated groups were clearly distinguished from the corn oil vehicle control group by all of the assays and by the effects of the modulators in three of the four assay/modulator combinations. Enzyme activities of the phenobarbital and saline groups were statistically similar ( P ≥0.05) when assayed without modulator added, but each assay/modulator combination distinguished between these groups. The PCB-treated group was distinguished from the corn oil vehicle control group only for BROD activity, with or without the presence of modulator. Graphing of per cent modulation of BROD activity versus initial BROD activity provided the clearest distinction between all of the study groups. Identification of these selective in vitro modulators may improve detection and measurement of low level cytochrome P450 induction in avian species. Also, both the monooxygenase activities induced and the impacts of the modulators indicated differences between mammalian and avian cytochromes P450.

Comparative Biochemistry and Physiology, Part C: P↗

USGS2018

Potential toxicity of complex mixtures in surface waters from a nationwide survey of United States streams: Identifying in vitro bioactivities and causative chemicals

While chemical analysis of contaminant mixtures remains an essential component of environmental monitoring, bioactivity-based assessments using in vitro systems increasingly play a role in the detection of biological effects. Historically, in vitro assessments focused on a few biological pathways, e.g., aryl hydrocarbon receptor (AhR) or estrogen receptor (ER) activities. High-throughput screening (HTS) technologies have greatly increased the number of biological targets and processes that can be rapidly assessed. Here we screened extracts of surface waters from nationwide survey of United States (US) streams for bioactivities associated with 69 different endpoints using two multiplexed HTS assays. Bioactivity of extracts from 38streams was evaluated and compared with concentrations of over 700 analytes to identify chemicals contributing to observed effects. Eleven primary biological endpoints were detected. Pregnane X receptor and AhR-mediated activities were the most commonly detected. Measured chemicals did not completely account for AhR and PXR responses. Surface waters with AhR and PXR effects were associated with low intensity, developed land-cover. Likewise, elevated bioactivities frequently associated with wastewater discharges included endocrine related endpoints— ER and glucocorticoid receptor (GR). These results underscore the value of bioassay-based monitoring of environmental mixtures for detecting biological effects that could not be ascertained solely through chemical analyses.

Environmental Science & Technology↗

USGS2022

Spatial distribution and physicochemical properties of respirable volcanic ash from the 16-17 August 2006 Tungurahua eruption (Ecuador), and alveolar epithelium response in-vitro

Tungurahua volcano (Ecuador) intermittently emitted ash between 1999 and 2016, enduringly affecting the surrounding rural area and its population, but its health impact remains poorly documented. We aim to assess the respiratory health hazard posed by the 16–17 August 2006 most intense eruptive phase of Tungurahua. We mapped the spatial distribution of the health-relevant ash size fractions produced by the eruption in the area impacted by ash fallout. We quantified the mineralogy, composition, surface texture, and morphology of a respirable ash sample isolated by aerodynamic separation. We then assessed the cytotoxicity and pro-inflammatory potential of this respirable ash toward lung tissues in-vitro using A549 alveolar epithelial cells, by electron microscopy and biochemical assays. The eruption produced a high amount of inhalable and respirable ash (12.0–0.04 kg/m 2 of sub-10 μm and 5.3–0.02 kg/m 2 of sub-4 μm ash deposited). Their abundance and proportion vary greatly across the deposit within the first 20 km from the volcano. The respirable ash is characteristic of an andesitic magma and no crystalline silica is detected. Morphological features and surface textures are complex and highly variable, with few fibers observed. In-vitro experiments show that respirable volcanic ash is internalized by A549 cells and processed in the endosomal pathway, causing little cell damage, but resulting in changes in cell morphology and membrane texture. The ash triggers a weak pro-inflammatory response. These data provide the first understanding of the respirable ash hazard near Tungurahua and the extent to which it varies spatially in a fallout deposit.

GeoHealth↗

USGS2017

Osmoregulation and muscle water control in vitro facing salinity stress of the Amazon fish Oscar Astronotus ocellatus (Cichlidae)

Specimens of Oscar Astronotus ocellatus from a fish farm were abruptly submitted to salt stress of 14 ppt and 20 ppt, for 3 and 8 h to determine their plasma osmolality. Muscle wet body mass change in vitro was analyzed from control freshwater animals. Fish in 14 ppt presented no osmolality distress even after 8 h. In 20 ppt, a slight increase (10%) in plasma osmolality was observed for both times of exposure when compared to control fish. Muscle slices submitted in vitro to hyper-osmotic saline displayed decreased body mass after 75 min, and slices submitted to hypo-osmotic saline displayed increased body mass after 45 min when compared to control (isosmotic saline). These results reinforce A. ocellatus ’s euryhalinity. The fish were able to regulate its internal medium and tolerate 14 ppt, but presented an intense osmotic challenge and low muscle hydration control when facing salinities of 20 ppt.

Marine and Freshwater Behaviour and Physiology↗

USGS2006

Method for in vitro screening of aquatic fungicides

Methods were developed for in vitro screening of candidate aquatic fungicides for efficacy against Achlya flagellata, A. racemosa, Saprolegnia hypogyna and S. megasperma . Agar plugs containing fungal hyphae, removed from the edge of actively growing colonies, were placed in the depressions of spot plates containing 1.0, 10.0 and 100 mg/l of the candidate compounds for 15 or 60 min. After exposure, the plugs were transferred on to filter papers (0.45-μm pore) in a holder, rinsed, and then placed on cornmeal agar medium in tri-petri dishes. The plates were checked for mycelial growth after 48, 96 and 168 h of incubation in a lighted (400–800 μm) environmental control chamber at 20±2°C. Criteria for the acceptance or rejection of candidate aquatic fungicides for further study were based on the antifungal spectrum index (ASI) comparisons between respective compounds and malachite green after 48 h and the concentration level producing complete growth inhibition. Candidate compounds whose ASI was Jess than 50% that of malachite green after 48 h or did not inhibit growth at levels less than 100 mg/l were rejected. This method provides a base from, which in vivo and definitive test regimens can be developed. Preliminary in vitro screening of candidate fungicides reduces the need for costly in vivo tests on compounds that have low antifungal activity.

Journal of Fish Diseases↗

USGS2012

Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

Reproduction in Domestic Animals↗

USGS2013

In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

Background: Transmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids (chronic wasting disease; CWD). The geographical range of bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown. Results: We acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A 136 R 154 Q 171 ). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A 136 R 154 Q 171 genotype sheep. Conclusions: Our results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species.

BMC Veterinary Research↗

USGS2016

Combined exposure of diesel exhaust particles and respirable Soufrière Hills volcanic ash causes a (pro-)inflammatory response in an in vitro multicellular epithelial tissue barrier model

Background There are justifiable health concerns regarding the potential adverse effects associated with human exposure to volcanic ash (VA) particles, especially when considering communities living in urban areas already exposed to heightened air pollution. The aim of this study was, therefore, to gain an imperative, first understanding of the biological impacts of respirable VA when exposed concomitantly with diesel particles. Methods A sophisticated in vitro 3D triple cell co-culture model of the human alveolar epithelial tissue barrier was exposed to either a single or repeated dose of dry respirable VA (deposited dose of 0.26 ± 0.09 or 0.89 ± 0.29 μg/cm 2 , respectively) from Soufrière Hills volcano, Montserrat for a period of 24 h at the air-liquid interface (ALI). Subsequently, co-cultures were exposed to co-exposures of single or repeated VA and diesel exhaust particles (DEP; NIST SRM 2975; 0.02 mg/mL), a model urban pollutant, at the pseudo-ALI. The biological impact of each individual particle type was also analysed under these precise scenarios. The cytotoxic (LDH release), oxidative stress (depletion of intracellular GSH) and (pro-)inflammatory (TNF-α, IL-8 and IL-1β) responses were assessed after the particulate exposures. The impact of VA exposure upon cell morphology, as well as its interaction with the multicellular model, was visualised via confocal laser scanning microscopy (LSM) and scanning electron microscopy (SEM), respectively. Results The combination of respirable VA and DEP, in all scenarios, incited an heightened release of TNF-α and IL-8 as well as significant increases in IL-1β, when applied at sub-lethal doses to the co-culture compared to VA exposure alone. Notably, the augmented (pro-)inflammatory responses observed were not mediated by oxidative stress. LSM supported the quantitative assessment of cytotoxicity, with no changes in cell morphology within the barrier model evident. A direct interaction of the VA with all three cell types of the multicellular system was observed by SEM. Conclusions Combined exposure of respirable Soufrière Hills VA with DEP causes a (pro-)inflammatory effect in an advanced in vitro multicellular model of the epithelial airway barrier. This finding suggests that the combined exposure to volcanic and urban particulate matter should be further investigated in order to deduce the potential human health hazard, especially how it may influence the respiratory function of susceptible individuals (i.e. with pre-existing lung diseases) in the population.

Particle and Fibre Toxicology↗

USGS1995

Gonadal steroidogenesis in-vitro from juvenile alligators obtained from contaminated or control lakes

The ubiquitous distribution of many contaminants and the nonlethal, multigenerational effects of such contaminants on reproductive, endocrine, and immune systems have led to concerns that wildlife worldwide are affected. Although the causal agents and effects are known for some species, the underlying physiological mechanisms associated with contaminant-induced reproductive modifications are still poorly understood and require extensive research. We describe a study examining the steroidogenic activity of gonads removed from juvenile alligators (Alligator mississippiensis) obtained from contaminated or control lakes in central Florida. Synthesis of estradiol-17 beta (E2) was significantly different when ovaries from the contaminated and control lakes were compared in vitro. Additionally, testes from males obtained from the contaminated lake. Lake Apopka, synthesized significantly higher concentrations of E2 when compared to testes obtained from control males. In contrast, testosterone (T) synthesis from all testes examined in this study displayed a normal pattern and produced concentrations greater than that observed from ovaries obtained from either lake. Interestingly, the pattern of gonadal steroidogenesis differs from previously reported plasma concentrations of these hormones obtained from the same individuals. We suggest that the differences between the in vivo and in vitro patterns are due to modifications in the hepatic degradation of plasma sex steroid hormones.

Environmental Health Perspectives↗

USGS1989

Suppression of antibody-producing cells in rainbow trout spleen sections exposed to copper in vitro

Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri ) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with dinitrophenyl-Ficoll and cultured in Eagle's minimum essential medium with 2% fetal calf serum; half of the medium was withdrawn and replaced every other day. The passive hemolytic plaque assay was used to determine the number of antibody-producing cells 10 d after injection. In the sections cultured with the high copper concentration (100 μg/mL), all cells died; at copper concentrations of 0.1–10 μg/mL, leukocytes remained viable, but fewer antibody-producing cells were present than in organ sections cultured in medium without copper. This in vitro method reduces the number of animals needed and the length of time required to determine toxicity and immunosuppression, and it provides information on the effects of certain environmental pollutants on fish.

Journal of Aquatic Animal Health↗

USGS1992

In vitro infection of salmonid epidermal tissues by infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha ; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.

Journal of Aquatic Animal Health↗

USGS2015

Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrP C ) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrP C that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrP C in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats ( Lynx rufus ) may be susceptible to white-tailed deer ( Odocoileus virginianus ) chronic wasting disease agent.

Journal of Visualized Experiments↗

USGS2003

Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5'-bromo-2'-deoxyuridine

In this study we present a method for the measurement of in vitro mitogenesis in fish leucocytes that is based on the incorporation of the thymidine analogue 5′-bromo-2′-deoxyuridine (BrdU) into the DNA of replicating cells, followed by ELISA-based detection. This technique, adapted from methods developed for mammalian cells, operates on a similar biological principle to 3 H-thymidine incorporation, but circumvents the logistical and safety issues inherent with the radioactive label. Because it directly measures DNA proliferation, the assay has advantages over other colorimetric methods that may be strongly influenced by leucocyte metabolic status. Using BrdU incorporation followed by ELISA, we evaluate the responsiveness of rainbow trout ( Oncorhynchus mykiss [Walbaum]) leucocytes to the mammalian T-cell mitogen Concanavalin A (Con A) as well as the differential response of white perch ( Morone americana [Gmelin]) leucocytes to Con A and pokeweed mitogen. Specific considerations intrinsic to the assay system are discussed, including the implications of utilising enzyme-based detection.

Fish and Shellfish Immunology↗

USGS1997

Physiological levels of testosterone kill salmonid leukocytes in vitro

Adult spring chinook salmon (Oncorhynchus tshawytscha) elaborate high plasma concentrations of testosterone during sexual maturation, and these levels of testosterone have been shown to reduce the salmonid immune response in vitro. Our search for the mechanism of testosterone's immunosuppressive action has led to the characterization of an androgen receptor in salmonid leukocytes. In the present study we examined the specific effects that testosterone had on salmonid leukocytes. Direct counts of viable leukocytes after incubation with and without physiological levels of testosterone demonstrate a significant loss of leukocytes in cultures exposed to testosterone. At least 5 days of contact with testosterone was required to produce significant immunosuppression and addition of a 'conditioned media' (supernatant from proliferating lymphocytes not exposed to testosterone) did not reverse the immunosuppressive effects of testosterone. These data lead us to conclude that testosterone may exert its immunosuppressive effects by direct action on salmonid leukocytes, through the androgen receptor described, and that this action leads to the death of a significant number of these leukocytes.

General and Comparative Endocrinology↗

USGS2022

Assessing methods for mitigating fungal contamination in freshwater mussel in vitro propagation

Many freshwater mussel species are critically imperiled, and propagation is essential for species ‘recovery.’ Fungal contamination can negatively affect in vitro propagation of freshwater mussels; thus, we investigated methods of mitigating fungal contamination. Specifically, we tested the effect of medium replacement frequency and antifungal (Amphotericin B) concentrations on risk of fungal contamination and transformation success of two species, Alasmidonta heterodon and congener, Alasmidonta undulata . We observed a trend of increased contamination risk in treatment groups that received medium replacements every 1 or 2 days (vs. every 3 days), but this trend was not statistically significant. We found that contamination risk differed significantly across Amphotericin B concentrations. Although contamination severity reduced transformation success, we found no significant difference in transformation success based on medium replacement frequency. Amphotericin B concentration was negatively correlated with transformation success, suggesting toxicity at higher concentrations. Additionally, we identified the fungal pathogen ( Candida parapsilosis ), the first knowledge of this fungus during mussel culture. Our findings suggest that low contamination results in high probability for transformation success; however, there is a tradeoff in higher concentrations of Amphotericin B. This study offers new ideas for improving mussel propagation techniques that may better control fungal contamination and increase transformation success.

Atlantic slope↗

USGS2013

Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication

Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2′-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17β-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.

Antiviral Research↗

USGS2009

Application of in vitro extraction studies to evaluate element bioaccessibility in soils from a transect across the United States and Canada

In vitro bioaccessibility tests (IVBA) are inexpensive, physiologically-based extraction tests designed to estimate the bioaccessibility of elements along ingestion exposure pathways. Published IVBA protocols call for the testing to be done on the <250-??m fraction of soil, as these particles are most likely to adhere to the hands of children and be ingested. Most IVBA in the literature to date have been applied to soil samples from highly contaminated sites or to spiked samples, and relatively little work has been done to evaluate bioaccessibility of elements in a wide variety of uncontaminated 'background' soils. In 2004, the US Geological Survey and the Geological Survey of Canada sampled soils along north-south and east-west transects across the two countries to test and refine sampling and analytical protocols recommended for the planned soil geochemical survey of North America. Samples were collected at 220 sites selected randomly at approximately 40-km intervals. The focus of the investigation presented in this paper was twofold: (1) to begin to examine variations in bioaccessibility of As, Cd, Cr, Ni and Pb in a number of 'background' (i.e., unpolluted) soils from around North America and (2) to determine if there are significant differences that would preclude using the standard size fraction of <2 mm for extraction with a simulated gastric fluid as an expeditious and inexpensive bioaccessibility screening tool for the large numbers of future samples to be collected by this continental-scale project. A subset of 20 soil samples collected along the north-south transect at a depth of 0-5 cm was used for this study. Two separate size fractions (<2 mm and <250 ??m) were extracted using a simulated human gastric fluid consisting of a solution of HCl and glycine adjusted to a pH of 1.5. In general, the leachate results for the <2-mm size fraction were not substantially different than those for the <250-??m size fraction for concentrations of As, Cd, Cr, Ni and Pb. Leachate concentrations for Cd, Ni and Pb appear to be controlled to some extent by the total concentration of the element in soil. Bioaccessibility of the elements in this study decreased in the order, Cd > Pb > Ni > As > Cr.

Applied Geochemistry↗

USGS2024

In vitro impacts of glyphosate on manatee lymphocytes

Exposure to contaminants, such as the herbicide glyphosate, can suppress protective immune functions. Glyphosate is the herbicide most used worldwide and has been found in the plasma of more than 50 % of the Florida manatees ( Trichechus manatus latirostris ) and all-year-round in their aquatic environment. Our objectives were to analyze the consequences of glyphosate exposure on their immune responses via T-lymphocyte proliferation assays and transcriptomics. We isolated peripheral blood mononuclear cells (mainly lymphocytes) of free-ranging manatees and performed T-cell proliferation assays. We used transcriptomics to understand the consequences of glyphosate in vitro exposure. The three doses chosen ranged from environmentally relevant concentrations at 10 to 10,000 µg.L −1 that is considered an environmental contamination scenario. Glyphosate caused a dose-dependent reduction in T-lymphocyte proliferation, with a significant mean reduction of 27.3 % at 10,000 µg.L −1 and up to 51.5 % in some individuals. Additionally, T-lymphocyte proliferation was significantly reduced in mid-winter compared to early winter. Transcriptomic analysis of peripheral blood mononuclear cells indicated that all doses of glyphosate (10, 1,000, and 10,000 µg.L −1 ) resulted in up-regulation of genes related to acute phase inflammation and inhibition of the T-lymphocyte proliferation pathway. Exposure to this contaminant along with other environmental stressors, such as extreme winters and red tide, might further affect the adaptive immune response of this threatened species.

Florida↗