Stenocarpella maydis and its toxic metabolites: a South African perspective on diplodiosis

2015 ◽  
Vol 8 (3) ◽  
pp. 341-350 ◽  
Author(s):  
M.G. Masango ◽  
B.C. Flett ◽  
C.E. Ellis ◽  
C.J. Botha
Keyword(s):  
Cultured Cells ◽  
Histopathological Change ◽  
Test Methods ◽  
In Vitro Toxicity ◽  
Stalk Rot ◽  
Cytotoxicity Studies ◽  
Dehydrogenase Enzyme ◽  
Maize Stubble ◽  
Stenocarpella Maydis

Stenocarpella maydis is one of the most prevalent ear and stalk rot pathogens of maize globally, causing reductions of grain quality and yield. Various molecular methods, including polymerase chain reaction (PCR)-based techniques and nucleotide microarrays, have been developed for the identification of S. maydis infestation in maize grain. In addition to diplodiatoxin, new metabolites, namely dipmatol, diplonine and chaetoglobosins K and L, have been isolated from S. maydis infected cultures. S. maydis infected maize is also associated with intoxication in ruminants. Diplodiosis, a nervous disorder of cattle and sheep, results from ingestion of mouldy ears, kernels and maize stubble infected by S. maydis. Although this disease is most common in southern Africa, it has also been reported in Australia, Argentina and Brazil. Diplodiosis is characterised by reluctance of the animals to move, a wide-based stance, incoordination, paralysis and death. Myelin degeneration (status spongiosis) is the only histopathological change observed in affected animals, especially in cases of perinatal mortality. To date, none of the purified S. maydis metabolites has been administered to ruminants in order to reproduce diplodiosis. However, recent studies have focused on investigating the toxicity of the metabolites on cell cultures. Cytotoxicity studies where cultured cells were exposed to the S. maydis metabolites indicated that diplodiatoxin and dipmatol affected the activity of the mitochondrial succinate dehydrogenase enzyme and the overall viability of the cells. More detailed in vitro toxicity studies are still required to elucidate how the currently available S. maydis metabolites influence parameters such as the mechanism of cell death. Development of analytical test methods to quantify and establish the presence and distribution of these mycotoxins in infected maize commodities also needs investigation. It is also critical that the role of S. maydis stalk rot be evaluated as a potential source and cause of diplodiosis.

scholarly journals The in vitro assessment of the toxicity of volatile, oxidisable, redox-cycling compounds: phenols as an example

2021 ◽  
Author(s):  
Laia Tolosa ◽  
Teresa Martínez-Sena ◽  
Johannes P. Schimming ◽  
Erika Moro ◽  
Sylvia E. Escher ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Degradation Products ◽  
Degradation Kinetics ◽  
Redox Cycling ◽  
In Vitro Toxicity ◽  
Cross Contamination ◽  
Physicochemical Changes ◽  
In Vitro Assessment ◽  
Biological Performance

AbstractPhenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC–MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity.

Long-term In Vitro Toxicity Models: Comparisons Between a Flow-cell Bioreactor, a Static-cell Bioreactor and Static Cell Cultures

2002 ◽  
Vol 30 (5) ◽  
pp. 515-523 ◽  
Author(s):  
Patricia Pazos ◽  
Salvador Fortaner ◽  
Pilar Prieto
Keyword(s):  
Cell Culture ◽  
Cell Viability ◽  
Flow Cell ◽  
Model Systems ◽  
In Vitro Toxicity ◽  
Protein Determination ◽  
Efficient System ◽  
Cytotoxicity Studies

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse) and a static cell bioreactor system (CELLine CL 6-well), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl2; 10–7–10–8M) for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl2 concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl2. Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl2 concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.

Metal-induced hormesis requires cPKC dependent glucose transport and lowered respiration

2001 ◽  
Vol 20 (7) ◽  
pp. 347-358 ◽  
Author(s):  
L H Damelin ◽  
J J Alexander
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Mitochondrial Respiration ◽  
In Vitro Toxicity ◽  
Protein Kinase Activity ◽  
Energy Status ◽  
Independent Events ◽  
Cytotoxicity Studies

Previously, cytotoxicity studies using an 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT)based in vitro toxicity assay found that low concentrations of mercuric, cadmium and cupric chloride (0.7, 1 and 3 μM, respectively) induced hormesis in McCoy cells after 24 h exposure. An investigation of the biochemical events required for the induction of this phenomenon revealed that hormesis was dependent on two simultaneous but independent events, namely, an 11-15% conventional protein kinase C (cPKC)-dependent increase in glucose uptake and a protein synthesis-dependent 19-23% drop in mitochondrial respiration. The inhibition of either event was sufficient to abolish hormesis for all three metal toxicants. Furthermore, an investigation of the energy status of cells prior to and during hormesis revealed an oscillating level of ATP production found to be in phase with mitochondrial respiration, independent of cPKC activated glucose transport and found to coincide with a 16-20% drop in AMP-activated protein kinase activity. These findings suggest that hormesis is not a form of energy compensation but is most likely a reductive burst where an increase in glucose uptake together with a simultaneous reduction in oxygen consumption results in a significant increase in reduction equivalents, which may then be utilized by cells to counteract the effects of oxidative stress induced by heavy metal toxicants.

Hormesis: A stress response in cells exposed to low levels of heavy metals

2000 ◽  
Vol 19 (7) ◽  
pp. 420-430 ◽  
Author(s):  
L H Damelin ◽  
S Vokes ◽  
J M Whitcutt ◽  
S B Damelin ◽  
J J Alexander
Keyword(s):  
Stress Response ◽  
Cellular Response ◽  
Stress Proteins ◽  
Constitutive Expression ◽  
Rapid Test ◽  
Resistant Mutant ◽  
In Vitro Toxicity ◽  
Cytotoxicity Studies ◽  
Increased Activity

Cytotoxicity studies using a 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MT)-based in vitro toxicity assay revealed that McCoy cells exposed to low concentrations of mercuric (0.7 yM),cadmium (1 1M) and cupric chloride (3 tIM) exhibited significant increases in cellular activity. This increased activity, previously termed hormesis, coincided with the production ofhigh levels ofthe stress proteins, heat shock protein 70 (Hsp 70) and metallothionein, while the high constitutive expression of these proteins in cadmium-resistant mutant (CRM) cells corresponded to constitutive hormetic activity. Hormesis was found to obey uniform kinetics allowing for a mathematical description of this increased activity. These results suggest that hormetic activity is a specific cellular response, and most likely, a stress response to low but harmful levels of toxic agents and may therefore provide a rapid test for the presence of toxicants at concentrations associated with chronic toxicity.

Aquatic Toxicology in Vitro: A Brief Review

1994 ◽  
Vol 22 (4) ◽  
pp. 243-253
Author(s):  
Boris Isomaa ◽  
Henrik Lilius ◽  
Christina Råbergh
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Aquatic Organisms ◽  
Single Species ◽  
Screening Tests ◽  
Aquatic Toxicology ◽  
Toxicity Tests ◽  
In Vitro Toxicity ◽  
In Vitro Tests

There is an urgent need for effective in vitro tests in aquatic toxicology, because only a very small proportion of the chemicals in common use have been adequately tested for their toxicity to aquatic organisms and aquatic ecosystems. Toxicity tests with higher animals, besides being time-consuming and expensive, are ethically questionable, which further increases the importance of developing efficient in vitro toxicity tests. In developing in vitro tests for toxicity assessments, aquatic toxicology lags behind mammalian toxicology. Aqueous environmental chemistry is complex, and the sensitivity of the organisms living in a particular aquatic environment may vary considerably. The predictive value of single-species or cell culture tests is therefore generally considered to be low. Nevertheless, single-species tests, utilising bacteria, algae, protozoans and invertebrates, have frequently been used in in vitro toxicity studies of aquatic pollutants (mainly as screening tests). Attempts at large-scale validations are few. Such attempts seem to be hampered by the complexity of the aquatic ecosystem. Although cells from aquatic organisms have been isolated and cultured for many years, the use of isolated or cultured cells in aquatic toxicology has been limited. However, during the last few years, interest in the use of fish cells in toxicity testing has grown rapidly. For aquatic in vitro toxicology to develop further, a more comparative and mechanistic approach needs to be adopted.

scholarly journals Allozyme-Specific Modification of a Maize Seed Chitinase by a Protein Secreted by the Fungal Pathogen Stenocarpella maydis

2010 ◽  
Vol 100 (7) ◽  
pp. 645-654 ◽  
Author(s):  
Todd A. Naumann ◽  
Donald T. Wicklow
Keyword(s):  
Inbred Line ◽  
Fungal Pathogen ◽  
Genetic Basis ◽  
Inbred Lines ◽  
Ear Rot ◽  
Maize Seed ◽  
Stalk Rot ◽  
Stenocarpella Maydis ◽  
Commercial Field

Stenocarpella maydis causes both dry-ear rot and stalk rot of maize. Maize inbred lines have varying levels of resistance to ear rot caused by S. maydis. The genetic basis of resistance appears to rely on multiple genetic factors, none of which are known. The commonly used stiff-stalk inbred line B73 has been shown to be strongly susceptible to ear rot caused by S. maydis. Here, we report that the ChitA protein alloform from B73, ChitA-F, encoded by a known allele of the chiA gene, is susceptible to modification by a protein (Stm-cmp) secreted by S. maydis. We also identify a new allele of chiA (from inbred line LH82) which encodes ChitA-S, an alloform of ChitA that is resistant to Stm-cmp modification. Chitinase zymogram analysis of seed from a commercial field showed the presence of both ChitA alloforms in healthy ears, and showed that ChitA-F but not ChitA-S was modified in ears rotted by S. maydis. The ChitA-F protein was purified from inbred line B73 and ChitA-S from LH82. ChitA-F was modified more efficiently than ChitA-S by S. maydis protein extracts in vitro. The chiA gene from LH82 was cloned and sequenced. It is a novel allele that encodes six polymorphisms relative to the known allele from B73. This is the first demonstration that the susceptibility to modification of a fungal targeted plant chitinase differs among inbred lines. These findings suggest that the LH82 chiA allele may be a specific genetic determinant that contributes to resistance to ear rot caused by S. maydis whereas the B73 allele may contribute to susceptibility.

scholarly journals Large scale and integrated platform for digital mass culture of anchorage dependent cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kyoung Won Cho ◽  
Seok Joo Kim ◽  
Jaemin Kim ◽  
Seuk Young Song ◽  
Wang Hee Lee ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Mass Culture ◽  
Large Scale ◽  
Cultured Cells ◽  
Toxicity Testing ◽  
Industrial Applications ◽  
In Vitro Toxicity ◽  
Scale Production

Abstract Industrial applications of anchorage-dependent cells require large-scale cell culture with multifunctional monitoring of culture conditions and control of cell behaviour. Here, we introduce a large-scale, integrated, and smart cell-culture platform (LISCCP) that facilitates digital mass culture of anchorage-dependent cells. LISCCP is devised through large-scale integration of ultrathin sensors and stimulator arrays in multiple layers. LISCCP provides real-time, 3D, and multimodal monitoring and localized control of the cultured cells, which thereby allows minimizing operation labour and maximizing cell culture performance. Wireless integration of multiple LISCCPs across multiple incubators further amplifies the culture scale and enables digital monitoring and local control of numerous culture layers, making the large-scale culture more efficient. Thus, LISCCP can transform conventional labour-intensive and high-cost cell cultures into efficient digital mass cell cultures. This platform could be useful for industrial applications of cell cultures such as in vitro toxicity testing of drugs and cosmetics and clinical scale production of cells for cell therapy.

Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells

1999 ◽  
Vol 18 (3) ◽  
pp. 180-187 ◽  
Author(s):  
H Mirto ◽  
M P Barrouillet ◽  
M H Hengé-Napoli ◽  
E Ansoborlo ◽  
M Fournier ◽  
...  
Keyword(s):  
Neutral Red ◽  
Uranyl Acetate ◽  
In Vitro Toxicity ◽  
Transmission Microscopy ◽  
Morphological Studies ◽  
Cytotoxicity Studies ◽  
Chemical Conditions ◽  
Few Data ◽  
Electron Transmission Microscopy

Very few data are available concerning the in vitro toxicity of uranium. In this work, we have determined the experimental chemical conditions permitting the observation of uranium(VI) cytotoxicity on LLC-PK1 cells. Uranium solutions made either by dissolving uranyl acetate or nitrate crystals, or by complexing uranium with bicarbonate, phosphate or citrate ligands, were prepared and tested. Experiments demonstrated that only uranium solutions containing citrate and bicarbonate ligands concentrations tenfold higher than the metal, were soluble in the cell culture medium. Cytotoxicity studies of all these uranium compounds were performed on LLC-PK1 cells and compared using LDH release, neutral red uptake and MTT assays. Dose dependent cytotoxicity curves were only obtained with uranium-bicarbonate medium. This study has revealed a toxicity of uranium-bicarbonate complexes for 24 h expositions and for concentrations ranging from 7×10-4- 10-3 M, under these conditions, the CI50 (cytotoxicity index) was evaluated between 8.5 and 9×10-4 M. In contrast, we noticed a lack of cytotoxicity response for uranium(VI)-citrate complexes. Electron transmission microscopy studies revealed, when LLC-PK1 cells were exposed to the uranium-bicarbonate system, that uranium penetrated and precipitated within the cytoplasmic compartment. Morphological studies conducted with citrate complexes did not show any cellular intake of uranium.

scholarly journals Cytotoxicity Studies on Naproxen and Piroxicam Nanoformulations

2020 ◽  
Vol 8 (3) ◽  
pp. 87-94
Author(s):  
Sandeep Patnaik ◽  
K Madhusudhana Rao ◽  
Vijay Sai
Keyword(s):  
Cell Culture ◽  
Cell Viability ◽  
Cultured Cells ◽  
In Vitro Models ◽  
In Vivo Studies ◽  
Tetrazolium Salt ◽  
Mucous Layer ◽  
Cytotoxicity Studies

Caco-2 cells were used as in vitro models to assess the cell viability characteristics of the carriers Soluplus®, Gelucire 50/13 and PVP K25 and the nanoformulations of Naproxen and Piroxicam. The assessment of cell viability was done using the tetrazolium salt based MTT assay. Gelucire 50/13 and its NFs were observed to have slightly higher cytotoxicity than PVP and Soluplus® and their respective NFs. All the NFs were observed to follow the cytotoxicity trend of the polymers. Our results show that no significant decrease in cell viability was seen until 0.01% concentration of Gelucire 50/13 for 12-h exposure. The NFs as well as the polymers alone had no significant effect on the viability of Caco-2 cells below 0.01% concentrations. The intestine has a protective mucous layer, whereas the cell culture monolayers do not. The intestinal tissues also have more capacity to recover from trauma than the cultured cells. Hence the present NFs can be expected to show lesser cytotoxicity when subjected to in vivo studies.  

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