thiols and sulfur


This post will focus on two aspects of glyphosate:

alarmist theories[1,2]

In 2017 A Samsel (consulting biologist) and A Seneff (computer science professor at MIT) published a review type article showing temporal correlations between use of glyphosate containing Round Up and a myriad of diseases. They failed however, to make a convincing argument that there was a causal relationship based on scant biochemical studies. [1] Two years later Robin Mesnage and Michael Antoniou of Guy’s College in London attacked the logic behind the and Samsel and Seneff publication. [2]

Glyphosate reduces free thiols in proteins [3]

Background tidbits

Ford and coauthors of UC Berkeley are going to tell us that glyphosate metabolite glyoxylate poisons rat liver fatty acid metabolism associated enzymes. [3]

The glyoxylate cycle in plants is used for gluconeogenesis and allows the seed to use portions of the TCA cycle to convert fats to glucose. Note the two CO2 that are NOT released when glyoxylate bypasses this part of the TCA cycle.

The point of this cycle is to convert fats as they enter the TCA cycle as acetylCoa to glucose without losing any carbons to CO2. Mammals are generally considered to not utilize this cycle. An antibody technique called Western blotting was used in 1992 to detect enzymes of the glyoxylate pathway in human liver homogenates. [4]

the hepatic metabolites of glyphosate

Ford and coauthors i.p injected mice with 13C/ 15N]glyphosate (200 mg/kg 1x per day for 7 days) after which the livers were harvested and subjected to mass spectrometry techniques used to identify the modified peptides of proteins.

Red oxygens denote a partial negative charge. Ford and coauthors argue that thiol groups of cysteines form covalent bonds with the carbon that has only an “H”.
If relative amounts of 15N AMPA are set at 100, 25x that amount of glyphosate was never metabolized and only one tenth was glyoxylate. When looking at total glyoxylate levels, dosing with glyphosate results in a 3x increase in glyoxylate.

Ford and coauthors reacted free thiols with an iodoacetamide alkyne derivative. Loss of the probe on a thiol means something else has reacted with the cyseine thiol first. The alkyne was reacted with avidin that was used to enrich the proteins with streptavidin.

Image link: “Thiol modifications caused by ROS. Protein thiols react with ROS. The initial oxidation product of this reaction is sulfenic acid.

This transient modification may be stabilized by the protein microenvironment or condense with a second cysteine resulting in intramolecular (①) or intermolecular (②) protein disulfides. Sulfenic acid can also react with low molecular weight thiolreductants, such as GSH (③). Alternatively, sulfenic acids may be oxidized to sulfinic acid and under severe oxidizing conditions, sulfonic acid.” As revolutionary as the Ford study is, we just need to be realistic of other side reactions besides blocking of free thiols with glyoxylate.

Abbreviated results of fig 2…

The bigger the black bar is than the blue, the more formerly free cysteine that were unable to react with the probe.

A truncated table. Note that when blue bars are much smaller than black, the presumption is that that thiol has been modified by glyoxylate.

Acaa2 In the production of energy from fats, this is one of the enzymes that catalyzes the last step of the mitochondrial beta-oxidation pathway, an aerobic process breaking down fatty acids into acetyl-CoA (Probable). Using free coenzyme A/CoA, catalyzes the thiolytic cleavage of medium- to long-chain unbranched 3-oxoacyl-CoAs into acetyl-CoA and a fatty acyl-CoA shortened by two carbon atoms (Probable). Also catalyzes the condensation of two acetyl-CoA molecules into acetoacetyl-CoA and could be involved in the production of ketone bodies (Probable). Also displays hydrolase activity on various fatty acyl-CoAs,.

“Active site” thiol targeting of fatty acid metabolizing enzymes of fig 3

Here the L:H ratio is the ratio of labeled thiols in the vehicle treated versus glyphosate treated. A ratio of two means that glyphosate took out half of the catalytic thiols.

Ford et al never defined what they meant by “active site.” A search for catalytic thiolate anions involved in acaa2 catalysis did not yield immediate results. The did repeat their experiments with just glyoxylate, alleviating the need for liver metabolism of glyphosate.

That they got more labeling with glyoylate than glyphosate argues against any thought that maybe glyphosate has some structural similarity with acetyl CoA. It should be pointed out that the sterol carrier protein scp2 is mitochondrial and catalyzes the following reaction in the backward direction: acetyl-CoA + hexadecanoyl-CoA = 3-oxooctadecanoyl-CoA + CoA. Scp2 may be found in mitochondria and peroxisomes.

Looking “in the grass” of fig 2

Does every post glyphosate, formerly free thiol come from some acyl CoA pathway protein? We must look in the grass of results presented in figure 3.

Some lesser targets of glyphosate in the rat liver. [3]
  • Senlenbp1 takes a big hit with glyphosate. This enzyme Widely expressed. Highly expressed in liver, lung, colon, prostate, kidney and pancreas. In brain, present both in neurons and glia (at protein level). Down-regulated in lung adenocarcinoma, colorectal carcinoma and ovarian cancer. Two-fold up-regulated in brain and blood from schizophrenia patients. When this protein is not functional, afflicted individuals have elevated methanethiol and dimethylsulfide from the microbiome.
  • GSz1 Probable bifunctional enzyme showing minimal glutathione-conjugating activity with ethacrynic acid and 7-chloro-4-nitrobenz-2-oxa-1, 3-diazole and maleylacetoacetate isomerase activity. Has also low glutathione peroxidase activity with t-butyl and cumene hydroperoxides (By similarity). Is able to catalyze the glutathione dependent oxygenation of dichloroacetic acid to glyoxylic acid.
  • Tubb2A, major protein of microtubules found especially in the brain.
  • Park7 is more popularly known as the Parkinson’s Disease protein that mediates autophagy of damaged mitochondria. Protein and nucleotide deglycase that catalyzes the deglycation of the Maillard adducts formed between amino groups of proteins or nucleotides and reactive carbonyl groups of glyoxals. Thus, functions as a protein deglycase that repairs methylglyoxal- and glyoxal-glycated proteins, and releases repaired proteins and lactate or glycolate, respectively. Deglycates cysteine, arginine and lysine residues in proteins, and thus reactivates these proteins by reversing glycation by glyoxals. Acts on early glycation intermediates (hemithioacetals and aminocarbinols), preventing the formation of advanced glycation endproducts (AGE) that cause irreversible damage. Also functions as a nucleotide deglycase able to repair glycated guanine in the free nucleotide pool (GTP, GDP, GMP, dGTP) and in DNA and RNA. Is thus involved in a major nucleotide repair system named guanine glycation repair (GG repair), dedicated to reversing methylglyoxal and glyoxal damage via nucleotide sanitization and direct nucleic acid repair. Also displays an apparent glyoxalase activity that in fact reflects its deglycase activity. Plays an important role in cell protection against oxidative stress and cell death acting as oxidative stress sensor and redox-sensitive chaperone and protease; functions probably related to its primary function.

Slc transporters in supplemental data, only see mitocondria ones

Ford and coworkers [3] were generous enough to supply number for all of the labeled proteins they identified. Unfortunately only two classes of mitochondrial Slc super family members were identified.

 protein description genecont mean  cont SEM Gly meanGly SEM  p value
Slc25a13 Calcium-binding mitochondrial carrier protein Aralar2Slc25a138.
Slc25a15 Mitochondrial ornithine transporter 1Slc25a154.
Slc25a20 Mitochondrial carnitine/acylcarnitine carrier proteinSlc25a207.
Slc25a4 ADP/ATP translocase 1Slc25a45.
Slc25a5 ADP/ATP translocase 2Slc25a533.718.827.73.70.3849745
Slc27a2 very long-chain acyl-CoA synthetaseSlc27a213.03.516.03.60.290420579
Slc27a5 Bile acyl-CoA synthetaseSlc27a532.36.724.79.00.265195006
Slc transporter data from the supplemental data

Slc13A1 and/or Slc26A1 may have been modified by glyphosate metabolite glycosate. They were never detected because the probe does not bind to free thiols in these transporters.

Ford et al 2017 parting words

It should be noted that this post is presenting the data in a direction other than the fatty acid metabolism direction that the authors originally intended.

Why some thiols are more prone to reaction with glyoxylate than others may depend on the micro environment as well as whether gyloxylate resembles normal substrates. The mitochondria protein preferring nature of the probe may have been a factor. This statement is not meant in any way to devalue any of the enormous, and long overdue, insights into glyphosate toxicity.

Back to sulfate, Slc26A1 and autism [5-7]

A previous post on the sodium sulfate cotransporter Slc13A1 made mention of its partner Scl26A1. In this post two blood bicarbonate anions were the anions exchanged for sulfate. A recent study of the hepatic cell line HepG2 demonstrated that glyoxylate was not only a substrate for Slc26A1 but could also increase its expression. [5] A commentary on this publication suggested a link between renal conservation of sulfate and liver use of sulfate for detoxifying compounds like acetaminophen by sulfation. [6]

Depending on whether one is looking at hepatocytes or renal tubule epithelial cells, these transporters work together.

A 2012 report out of Poland suggested that autistic children have higher levels of oxalate in their blood plasma as well as their urine. [7] While there might be cytochrome P450s in the kidney to convert glyphosate to glyoxylate, inhibitory cysteine adducts of Slc26A1 would help explain high levels of oxalate in the plasma and slow sulfate in the plasma. We still need an explanation for high oxalate in the urine.

The sequence of human Slc26A1. We do not have a crystal structure of this transporter so it is hard to say how many of these cysteines are in disulfide bonds.

Samsel and Seneff presented some compelling and inconclusive correlations between use of glyphosate and many chronic diseases of modern times. [1] Mesnage and Antoiniou attacked their logic with the concern that glyphosate might be doing something good for the food industry. [2] Ford and authors presented a very nice hypothesis that glyphosate causes problems by virtue of thiol reactivity of its metabolite glycosate. [3] Perhaps some caution is warranted in that we have evidence that glyoxylate might be produced in the human liver. [4] We know that glyoxylate can be transported by the sulfate/oxalate exchanger [5] in ways that are important for liver and kidney function. [5, 6] We still have some unanswered questions as to whether this mechanism might explain some observations sulfate and oxalate [7] metabolism in autism.


  1. Samsel A, Seneff S. Glyphosate, pathways to modern diseases III: Manganese, neurological diseases, and associated pathologies. Surg Neurol Int. 2015 Mar 24;6:45. PMC free article
  2. Mesnage R, Antoniou MN. Facts and Fallacies in the Debate on Glyphosate Toxicity. Front Public Health. 2017 Nov 24;5:316. PMC free article
  3. Ford B, Bateman LA, Gutierrez-Palominos L, Park R, Nomura DK. Mapping Proteome-wide Targets of Glyphosate in Mice. Cell Chem Biol. 2017 Feb 16;24(2):133-140. free article
  4. Davis WL, Goodman DB. Evidence for the glyoxylate cycle in human liver. Anat Rec. 1992 Dec;234(4):461-8. PubMed
  5. Schnedler N, Burckhardt G, Burckhardt BC. Glyoxylate is a substrate of the sulfate-oxalate exchanger, sat-1, and increases its expression in HepG2 cells. J Hepatol. 2011 Mar;54(3):513-20. PubMed
  6. Stieger B. Regulation of the expression of the hepatocellular sulfate-oxalate exchanger SAT-1 (SLC26A1) by glyoxylate: a metabolic link between liver and kidney? J Hepatol. 2011 Mar;54(3):406-7. free article
  7. Konstantynowicz J, Porowski T, Zoch-Zwierz W, Wasilewska J, Kadziela-Olech H, Kulak W, Owens SC, Piotrowska-Jastrzebska J, Kaczmarski M. A potential pathogenic role of oxalate in autism. Eur J Paediatr Neurol. 2012 Sep;16(5):485-91. PubMed

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