Bartholomew Cubbins on Autism Episode 2: Waly et al, Mol Psych 2004
A rough discussion of Waly et al, Mol Psych 2004. Overall, the work is very interesting but the materials and methods description leaves a lot to be desired and the effect on PLM was marginal, often only 2-fold across several orders of magnitude of effector concentration. Further, many questions arise such as the form of the metals (counterion description?) and ensuring that the ionic strength and the pH remained constant across the effector titrations. What would be particularly interesting is comparing this assay (using maybe a mouse neuro line) with a side-by-side animal model experiment.
-BC
posted by Bartholomew Cubbins @ 1/12/2006 08:05:00 AM
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Mr Cubbins
I am very interested on your analysis. However, your links are not working in my PC. Do you have them for Quick time player?
Sorry if I disturbed you. If you can´t I will try again if I can download them.
María Luján Ferreira
Hi Maria,
if you right mouse click on the link you ought to get an option to download the file to your PC. I'm no expert on the compatibility of different versions of quicktime but that may be a problem. I encoded it in MPEG-4 using Quicktime in order to make it accessible to most people. The link is being reported as working for people in the US, but maybe bandwidth is an issue too. I don't know. Post back again if it doesn't work and I'll give you an URL to the video server.
-BC
Well, it did not work and I tried many times. It seems to be a bandwith problem or something like this because the PC is locked when begins to download the movie.
Do you have perhaps the movie to send it to me by e-mail or the URL?If you can´t do not worry about.
Thank you in advance
María Luján
To skirt any blogger-induced BW restrictions (I'm grasping for straws here) try:
http://blip.tv/file/9531
and
http://blip.tv/file/8273
If you're on dial-up, downloading video will not work no matter who is hosting the file. Hope that helps. I'm exploring using google video as a host, but at this point since you're the only one reporting problems, I'm out of ideas. Good luck.
-BC
Save the file and then rename it to something shorter like Deth_Match_2000.mov (make sure to keep the extension intact)QT has a problem with long filenames sometimes. I still haven't watched it but I have to first convert it to be viewed on my phone.
Ha ha - Deth_Match. A big thanks for the information that Deth is pronounced "deeth". I try not to mispronounce anyone's name given my own. "Deeth" was news to me.
-BC
Yeah but Deeth warmed over isn't as catchy ;-)
Hi Bartholomew
Unfortunately, I could not see the videos because some problem of compatibility. BTW, thank you clone3g for your advice. I could not save them either, because I had not the option.
However, I have some comments about this manuscript that perhaps you can be interested on, related to the use of the cell´s lines and the design of the experiments and , as it is usual with me now, with the questions :).Please let me know. Do you prefer I discuss with you here?
TIA
María Luján
Hi Maria, Fire away! Scientific conversation is cool, let's do it.
Hi
First, I think is interesting to look at the general attitude of the authors of this manuscript. They provide a lot of information about pathways/markers of potential involvement/dysfunction in ASD (MS, SAMe/SAH, dopamine activation of the D4 receptor). Figure 1 and Figure 7 shows the main biochemical pathways they are talking about. Based on their results on the SH-SY5Y cells they propose a series of targets of Hg and Al (MS- mainly) and their effects. They conclude that, under the limitations of their study,ethanol heavy metals and thimerosal interfere with MS activation and impair folate dependent methylation, that can be related to impared DNA methylation. Growth factors (Nerve growth factor-NGF, Brain derived neurotrophic factor-BDNF and Insuline growth factor 1-IGF-1 ) promote development of the neuronal phenotype and support the function and survival of differentiated neurons.
They include a comment about constraints or limitations of their work in page 11 about the use of SH-SY5Y cells –undifferentiated vs differentiated cells. They pointed clearly the need of more research in GF induced cellular differentiation. They pointed out the importance of MS but also other PI3 and MAP- kinase signaling pathways and how they may be affected by metal ions.
I have found several criticisms to these statements
1- One of the criticisms of this is how it is representing bioavailavility in vivo of Hg from thimerosal , that in vitro is full (comments form P. Offit and J. Golden in M. Psychyatry)
2- The use of neuroblastoma cells and therefore not normal differentiated cells.
Well, besides the accepted limitations of their study, I would propose to analyze under the following premises and questions the validity of this paper. I have researched about the value of all these so I have several manuscripts to analyze with you related to several of these questions. I do know that some manuscripts from Dr Haley for example are considered outdated- and I agree that they are, for example the “Baby haircuts..”. However, I think that in this field the analysis must be related also to what was the useful point you can find. There is no perfect scientific literature. So, for me, even if time demonstrated something to be inaccurate or even wrong, many ideas/manuscripts have their value in time, even if only pointed about some aspect that deserves more research. Remember Boor theory vs Quantum mechanics, for example. So, I respect all authors BUT consider that all manuscripts have very useful, useful and useless information, considering the evolution of the topic. The analysis of the relative weight of the three considerations in terms of useful information also will change with further research.
1-Autism is of genetic root in origin. Therefore we can say that ASD children will be different from birth. Are there any published papers about differences in ASD children in Growth factors-NGF, BDNF, IGF.1- in newborns, toddlers and adults?
2-Being of genetic root in origin and having enough published demonstration of structural differences in brains of autistic people vs normal people What is the best line of neuronal cells to analyze: susceptible ones or normal ones? Being of genetic root, the most similar cells would be not the normal ones, but the ones with some abnormality. If these SH-SY5Y cells represent the abnormality that can be present in autistic from birth is another point, but normal cells will not represent the nature of the neural cells in autistic children. Do you agree?
3-What is the published evidence about the bioavailability of thimerosal versus methyl mercury? Are there enough studies about what is the pathway that thimerosal follows when it is injected ? IT is known that the pharmacokinetics and dynamics of thimerosal is far from being enough studied or even understood clearly. It has been assumed different – and safer- than methyl mercury Is it so by recent published papers-last 5 years- besides the Burbacher study? For me Pichichiero data demonstrated only that thimerosal generates faster inorganic mercury than methyl mercury in blood.
4- What are the potential combination of genetics and these altered pathways that could give us some clue about the
a)crossing of the BBB by thimerosal /metabolites in autistic children
b) synergies with other components from vaccines , such as Al in the different biochemicalpathways Waly et al mentioned.
What are the published manuscripts in last years about genetics and heavy metal susceptibility?
Are there any published studies about the BBB permeability to heavy metals?
Are there any published studies about the possible chemical interaction between thimerosal and metabolites and Al (hydroxye and sulphate) metabolites to form in vivo complexes between them of different properties than the original compounds ?
I think that the real value of this paper would be (perhaps) more evident if all these related aspects are discussed.
Please let me know the pertinence of these questions before we begin
Do you want to Add/Clarify/change/discard questions? Change the approach?
I imagine this as the best approach but perhaps you consider there is a better one.
Please let me know
María Luján
Sorry, I typed with a mistake. Bohr Theory of hydrogen atom. I realized when I re-read.
MAría Luján
My issue with the format of this paper is that the materials and methods is sparse. These assays need to be clearly described.
Scientifically, I have problems with the following:
1. The authors apparently did not control for pH and ionic strength across the metal and drug titrations. It's not mentioned anywhere.
2. Were all the counterions similar for each metal? Again, not mentioned.
3. Does a 2-fold reduction in the IGF-1 enhancement due to thimerosal or metals mean anything? Is IGF-1 present at low doses already in autistic kids and if so what does a minute reduction in this already low concentration really mean?
4. How was the thimerosal handled? What are the quality control measures that were used? None are described.
5. Does an in vitro MS assay mirror what is seen in cell culture or in an animal model?
6. The type of cells - not a huge deal to me although I'm bothered that data for other cell lines isn't discussed - that's a weak point. The data could have been put in a supplementary datg file or something.
I hesitate to wade into the bioavailability issue since I have seen no convincing in vivo data showing active de/methylation of ethyl-and methyl-mercury. Further, I've never seen anything that convinces me that thimerosal breaks down into ethyl mercury in vivo. Maybe I've missed something but I doubt it. The experiment would be challenging but possible. If the sulfur in thimerosal was S35 then it could be tracked. Likewise one of the carbons could be C14 while the molecule could be tritiated on the other side of the sulfur to observe dissociation by comparing the C14/H3 ratio (should be 1 if it doesn't dissociate).
Anyway, those are some quick thoughts.
Hi Bartholomew
Well, I try to answer properly your questions.
1. The authors apparently did not control for pH and ionic strength across the metal and drug titrations. It's not mentioned anywhere.
Very good point. Well, I see in PLM the mention of Hank´s balanced salt solutions – a wash-( an attempt to generate some ionic salt adsorbed?). Here you can find the compositions of several Hank´s solution and the methodology of preparation.
http://zfin.org/zf_info/zfbook/chapt1/1.3.html
www.nature.com/bjp/journal/v130/n4/pdf/0703360a.pdf
Please see here how the pH of the Hank solution is mentioned.
For the MS activity, the pH mentioned is 7.4 from phosphate buffer.. Again the Hank´s solutions was cited. The reaction mixture section mentions pH 7.2.
But I agree with you that the authors had to clarify explicitly the reaction pHs they used.
2. Were all the counterions similar for each metal? Again, not mentioned.
In Table 1, Cu is cited as CuCl2 (Cu+2), CuCl (Cu+1), Hg as from HgCl2 (Hg+2) and Pb from Pb (NO3)2 (Pb+2). In fact, here is a mistake- for me- about the lack of the 2 for the NO3 in the table. Pb more stable oxidation states are +2 and +4 but +2 is thermodynamically favored (inert pair effect). So the counterions of Cu+2, Cu+and Hg+2 are chloride. PbCl2 is insoluble so they selected nitrate as counterion because this salt is a soluble one.
But it is certain that in the materials section this is not mentioned.
3. Does a 2-fold reduction in the IGF-1 enhancement due to thimerosal or metals mean anything?
I think that the use of IC50 is related to the relative concentrations to achieve them- please tell me if I am understanding well to you. So for
Hg+2 15 nM
Pb+2 100 nM
Al+3 biphasic with 0.1 and 200 nM
Cu+2 was detected as an exception
So in order of inhibitory strength to IC50 is Hg+2>Pb+2>Al+3, in the conditions of the essay.
Is IGF-1 present at low doses already in autistic kids and if so what does a minute reduction in this already low concentration really mean?
This manuscript reported that IGF-1 in the patients tested was lower than controls. If the reduction was enough to be compared in a straight form to the results of Waly et al. is difficult because this is in vivo, but I think that is a good point.
Dev Med Child Neurol. 2001 Sep;43(9):614-6.
Low levels of insulin-like growth factor-I in cerebrospinal fluid in children with autism.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11570630&query_hl=35&itool=pubmed_docsum
Another
J Child Neurol. 2003 Oct;18(10):693-7.
Neurotrophic factors in the pathogenesis of Rett syndrome.
Riikonen R.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14649551&query_hl=35&itool=pubmed_docsum.
4. How was the thimerosal handled? What are the quality control measures that were used? None are described.
I could not find anything explicitly such as quality control measures but in figure 5 different concentrations are cited for example 10 micro Molar in b) , in c)to f) 1 microMolar for thimerosal. I imagine it was handled as solutions as in the case of the other metal salts solutions. Again here I found a mistake because PbCl2 is insoluble, depending on concentration (Kps 298K=2.4 x 10-4). For me here there must be Pb(NO3)2, even if the concentration is so low. In table 1 is included as Pb(NO3)2.
5. Does an in vitro MS assay mirror what is seen in cell culture or in an animal model?
You can appreciate the difficult issue of methionine synthase structure and function in
http://www.pnas.org/cgi/content/full/101/11/3729
Also, it is very interesting that Zn is involved in the function of this enzyme.
Inorg Chem. 2000 Sep 18;39(19):4347-53.
Synthetic models for the zinc sites in the methionine synthases.
Chiou SJ, Innocent J, Riordan CG, Lam KC, Liable-Sands L, Rheingold AL.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11196931&query_hl=57&itool=pubmed_docsum
Curr Opin Chem Biol. 1997 Oct;1(3):332-9.
Enzyme-catalyzed methyl transfers to thiols: the role of zinc.
Matthews RG, Goulding CW.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9667865&query_hl=58&itool=pubmed_docsum
In this page I found a very graphic explanation about sustitution of Zn by Hg /Cd that could explain the molecular mechanism of MS blocking by heavy metals.
http://www.uvm.edu/~swgordon/231-03/finprojects/Kay/KAYINOR.HTM
so going to more basic science…
Matzapetakis, M.; Farrer, B. T.; Weng, T.-C.; Hemmingsen, L.; Penner-Hahn, J. E.; Pecoraro, V. L.; " Comparison of the Binding of Cadmium(II), Mercury(II), and Arsenic(III) to the de Novo Designed Peptides TRI L12C and TRI L16C" J. Am. Chem. Soc., 2002, 124(27) 8042-8054
6. The type of cells - not a huge deal to me although I'm bothered that data for other cell lines isn't discussed - that's a weak point. The data could have been put in a supplementary datg file or something.
I think that the same work could be done with normal cells vs neuroblastome cells (considered these as a model to clarify of cells in developmental problems)
Well, about bioavailability of thimerosal and metabolic pathway, I found this
Anal Chem. 2003 Aug 15;75(16):4120-4.
Determination of methylmercury, ethylmercury, and inorganic mercury in mouse tissues, following administration of thimerosal, by species-specific isotope dilution GC-inductively coupled plasma-MS.
Qvarnstrom J, Lambertsson L, Havarinasab S, Hultman P, Frech W.
Analytical Chemistry, Department of Chemistry, Umea University, S-901 87 Umea, Sweden. johanna.qvarnstrom@chem.umu.se
Isotopically enriched HgO standards were used to synthesize CH3(200)Hg+ and C2H5(199)Hg+ using Grignard reagents. These species were employed for isotope dilution GC-ICPMS to study uptake and biotransformation of ethylmercury in mice treated with thimerosal, (sodium ethylmercurithiosalicylate) 10 mg L(-1) in drinking water ad libitum for 1, 2.5, 6, or 14 days. Prior to analysis, samples were spiked with aqueous solutions of CH3(200)Hg+, C2H5(199)Hg+, and 201Hg2+ and then digested in 20% tetramethylammonium hydroxide and extracted at pH 9 with DDTC/toluene. Extracted mercury species were reacted with butylmagnesium chloride to form butylated derivatives. Absolute detection limits for CH3Hg+, C2H5Hg+, and Hg2+ were 0.4, 0.2, and 0.6 pg on the basis of 3sigma of five separate blanks. Up to 9% of the C2H5Hg+ was decomposed to Hg2+ during sample preparation, and it is therefore crucial to use a species-specific internal standard when determining ethylmercury. No demethylation, methylation, or ethylation during sample preparation was detected. The ethylmercury component of thimerosal was rapidly taken up in the organs of the mice (kidney, liver, and mesenterial lymph nodes), and concentrations of C2H5Hg+ as well as Hg2+ increased over the 14 days of thimerosal treatment. This shows that C2H5Hg+ in mice to a large degree is degraded to Hg2+. Increased concentrations of CH3Hg+ were also observed, which was found to be due to impurities in the thimerosal.
I included the full explanation because I think is the first study of degradation in vivo I know enough complete. Do you know about another?
What about methylation in vivo of Hg+2 so transformation to ... methylmercury? This manuscript pointed the presence of Hg+2 (inorganic) from thimerosal breakdown I know this is ONE study, but it is important: thimerosal can (please see my emphasis can)
a) remain as thimerosal (how much??)
b) converted to Hg+2, following Pichichiero, this happens after vaccination.
c) converted to methylHg by in vivo methylation, from Hg+2 (how much??)
d) after b and c, react with Al compounds ( if we consider vaccines, they include Al as sulphate and hydroxide) doing complexes in vivo ( how much??),
I wonder, who is exploring this possibility, in genetically different children from birth, considering the overall vaccination schedule??
Only asking and proposing routes. I have my opinion, but is personal. I do think we need more studies about all this.
A Textbook of Modern Toxicology from E. Hogdson, 2004, has a huge amount of Gene interactions and xenobiotics , very interesting. Please let me know if you are interested (I have the .pdf)
In Kev´s forum I included the Clarkson paper and several references about neuroimmunotoxicology.
Sorry by the LOOOONG post by I feel that I must try to comment the best I can the points.
I have more information if you are interested on other aspects (BBB and heavy metals, BBB and MCP-1, other aspects of heavy metals and environmetal insult etc) Please contact me if you are interested on the discussion of this material.
Sincerely María Luján
Re: bioavailablity:
I think that the sweden study is a good attempt, but the issue I saw when I first read the paper (some time ago so I should go back and revisit it) was a lack of control for the possibility of thimerosal degradation in the water bottle. I have all the confidence in the world with the competency of that group, it is just that I saw that as a potential hole.
Re: metals and concentrations in Waly et al.
When I want to get a metal into solution I drop it into acid, usually HCl. I have a tough time believing that the metals used in this study were all neutral pH. As you pointed out, some metals are extremely insoluble and take a very low pH to get into solution. So my question is whether some of the results can be viewed as a pH effect? Further, were all the metals chloride versions or were other counterions present - could this be an issue? Also, titrating in a divalent (Cu++) changes the ionic strength by the square of the concentration whereas the ionic strength changes linearly with monovalent (Cu+). Is there an ionic strength component to the response? This could have been accounted for using a mixture of varying concentrations of metal "x" with the testing metal in order to keep total ionic strength constant.
It's stuff like this that really bothers me about the paper. I'm not going to argue that dropping metal onto cells isn't going to ellicit a response - of course it will. What I'm talking about is enacting careful experimental protocol.
I know enough about you and your knowledge of chemistry to know that if you had done this experiment you would have controlled for all these things and would have reported the important details. I hope I would have.
BTW, I need to do something with the format of this blogger template. It is exceedingly difficult to read a comment longer than 2 sentences.
Hi Bart ( can I call you so?)
Well, from what I read the Sweden group tested the degradation and they found no a lot of degradation, but a bit (near 9 %, before injection).
Up to 9% of the C2H5Hg+ was decomposed to Hg2+ during sample preparation...No demethylation, methylation, or ethylation during sample preparation was detected.
I wonder for example in vaccines if the thimerosal is ALL as such or it can be degradated partially I never saw a test of thimerosal/EtHg/inorg Hg done to vaccines Did you? Thank you, I never thought in that possibility before, but it is, because there are a lot of time between the manufacture and the use of the vaccine and a lot of possible interactions ( with Al compounds in the bottle?).
What I saw from the use of Hank´s solutions is that HCl is included.
Ionic strength is a good point, but I imagine that, if they use Hank´s solutions they would have to consider all the concentrations. In some way, the ion concentration is very low (uM)perhaps in comparison to the concentrations of the compounds used in the Hank´s solution. Generally, buffer compounds have high concentrations because what it matters is the relation from Anion to non-ionizated form and they can increase their buffer capacity with higher concentrations.They would have to calculate and to report the ionic strengths and control if it is the same. But I think that, if all the amounts of solutions is maintained, the main contribution would not be from the ion concentrations but form the Hank´s solutions Don´t you think?
Thank you for your comment about me. I always do my best when I must report my research results... but we are all humans and the possibility of unnoticed mistakes is always there, even if these mistakes in particular can be avoided being enough careful.
I am very sorry. It was my mistake. Generally, I use to make long posts/explanations. I hope this did not bother you.
I maintain my offer to discuss further information, here, in Kev´s forum or by email. I think that I have not the possibility of external comment validation/discussion of what I can research/think how can I learn?
As I always say, I am a student of Autism and of my son´s autism so I would appreciate your help. But if you are not interested it is OK.Please do not feel pressed anyway.
TIA
MAría Luján
Bart, BC, hey jerkee - the names don't matter much to me.
Your point about total ionic strength is right on - I agree but I wish that (especially given the modest changes in IGF-1 activation) it was controlled for.
I think it far more likely that the pH was changed over the course of the titration in Waly et al. Maybe I'm wrong, but they could have cleared that up with one sentence in the materials/methods.
So I've gone into the swedish paper and I cannot find where the group tested the integrity of thimerosal while sitting inside the water bottle. In short, were the mice drinking thimerosal-water by day 5 or were they drinking Et-Hg in water by that point? They absolutely tested their samples upon preparing them, but I see the achilles heel being the water bottle "incubation" and its impact upon thimerosal.
Maybe I should do this paper as a quicktime too.
Hi Bart
Your statement is true about the needed control of thimerosal integrity and also about the need of clarification of the pH.
Again, I wonder what has been the control about the thimerosal integrity in vaccines?
When the vaccine is finally used. what is being injected really as such but a mix of -thimerosal, EtHg and potential complexes with Al/others?? Who tested the thimerosal degradation/interaction with other compounds present in vaccines in time?
Also we must remember that this mice were given drinking water and thimerosal was not injected, so the pathway is different, injected or ingested, so Do you remember my post in Kevin´s forum about? I am concerned about this. When ingested, the delivery is supposed to be different.
María Luján
Good points and I wish I knew the answers.
Hi
Perhaps you can be interested on this manuscript on tranport of metals in vivo
Environ Health Perspect. 2002 Oct;110 Suppl 5:689-94.
http://www.pubmedcentral.gov/picrender.fcgi?artid=1241226&blobtype=pdf
Transport of toxic metals by molecular mimicry.
Ballatori N.
Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642, USA. Ned_Ballatori@urmc.rochester.edu
Intracellular concentrations of essential metals are normally maintained within a narrow range, whereas the nonessential metals generally lack homeostatic controls. Some of the factors that contribute to metal homeostasis have recently been identified at the molecular level and include proteins that mediate import of essential metals from the extracellular environment, those that regulate delivery to specific intracellular proteins or compartments, and those that mediate metal export from the cell. Some of these proteins appear highly selective for a given essential metal; however, others are less specific and interact with multiple metals, including toxic metals. For example, DCT1 (divalent cation transporter-1; also known as NRAMP2 or DMT1) is considered to be a major cellular uptake mechanism for Fe(2+) and other essential divalent metals, but this protein also mediates uptake of Cd(2+), Pb(2+), and possibly of other toxic divalent metals. The ability of nonessential metals to interact with binding sites for essential metals is critical for their ability to gain access to specific cellular compartments and for their ability to disrupt normal biochemical or physiological functions. Another major mechanism by which metals traverse cell membranes and produce cell injury is by forming complexes whose overall structures mimic those of endogenous molecules. For example, it has long been known that arsenate and vanadate can compete with phosphate for transport and metabolism, thereby disrupting normal cellular functions. Similarly, cromate and molybdate can mimic sulfate in biological systems. Studies in our laboratory have focused on the transport and toxicity of methylmercury (MeHg) and inorganic mercury. Mercury has a high affinity for reduced sulfhydryl groups, including those of cysteine and glutathione (GSH). MeHg-l-cysteine is structurally similar to the amino acid methionine, and this complex is a substrate for transport systems that carry methionine across cell membranes. Once MeHg has entered the cell, some of it binds to GSH, and the resulting MeHg-glutathione complex appears to be a substrate for proteins that mediate cellular export of glutathione S-conjugates, including the apically located MRP2 (multidrug resistance-associated protein 2) transporter, a member of the adenosine triphosphate-binding cassette protein superfamily. Because other toxic metals also form complexes with endogenous molecules, comparable mechanisms may be involved in their membrane transport and disposition.
Other
Toxicol Appl Pharmacol. 2004 Feb 15;195(1):73-82.
Development of an in vitro blood-brain barrier model-cytotoxicity of
mercury and aluminum.
Toimela T, Maenpaa H, Mannerstrom M, Tahti H.
University of Tampere, Medical School, 33014 University of Tampere,
Tampere, Finland. Tarja.Toimela@...
In this study, in vitro blood-brain barrier (BBB) models composed of
two different cell types were compared. The aim of our study was to
find an alternative human cell line that could be used in BBB
models. Inorganic and organic mercury and aluminum were studied as
model chemicals in the testing of the system. BBB models were
composed of endothelial RBE4 cell line or retinal pigment epithelial
(RPE) cell line ARPE-19 and neuronal SH-SY5Y cells as target cells.
Glial U-373 MG cells were included in part of the tests to induce
the formation of a tighter barrier. Millicell CM filter inserts were
coated with rat-tail collagen, and RBE4 or ARPE-19 cells were placed
on the filters at the density of 3.5-4 x 10(5) cells/filter. During
culture, the state of confluency was microscopically observed and
confirmed by the measurement of electrical resistance caused by the
developing cell layer. The target cells, SH-SY5Y neuroblastoma
cells, were plated on the bottom of cell culture wells at the
density of 100000 cells/cm(2). In part of the studies, glial U-373
MG cells were placed on the under side of the membrane filter. When
confluent filters with ARPE-19 or RBE4 cells were placed on top of
the SH-SY5Y cells, different concentrations of mercuric chloride,
methyl mercury chloride, and aluminum chloride were added into the
filter cups along with a fluorescent tracer. Exposure time was 24 h,
after which the cytotoxicity in the SH-SY5Y cell layer, as well as
in the ARPE-19 or RBE4 cell layer, was evaluated by the luminescent
measurement of total ATP. The leakage of the fluorescent tracer was
also monitored. The results showed that both barrier cell types were
induced by glial cells. Inorganic and organic mercury caused a
leakage of the dye and cytotoxicity in SH-SY5Y cells. Especially,
methyl mercury chloride could exert an effect on target cells before
any profound cytotoxicity in barrier cells could be seen. Aluminum
did not cause any leakage in the barrier cell layer, and even the
highest concentration (1 mM) of aluminum did not cause any
cytotoxicity in the SH-SY5Y cells. In conclusion, BBB models
composed of RBE4 and ARPE-19 cells were able to distinguish between
different toxicities, and ARPE-19 cells are thus promising
candidates for studies of drug penetration through the blood-brain
barrier.
María Luján
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