Glutathione Conjugation

This page contains educational material primarily about Chronic Inflammatory Response Syndrome due to a water damaged building (CIRS). This is a biotoxin illness that is caused by the interaction between mold, bacteria and chemicals used in building materials. Other biotoxin illnesses have similar signs and symptoms. They are also treated similarly. Some of them are discussed on this site. Additionally, there is imformation on this site about Nutrigenomics and Biotransformational pathways as you really can't treat someone with a biotoxin illness without including analysis and treatment of these pathways. This information is for educational purposes only. Nothing in this text is intended to serve as medical advice. All medical decisions should be made only with the guidance of your own personal medical authority. I am doing my best to get this data up quickly and correctly. If you find errors in this data, please let me know.

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For an in depth description of glutathione and how it is made, recycled and things that can assist in glutathione support, (see more data on this important nutrient here). On this page we discuss what glutathione does in relation to Phase II glutathione conjugation.

Glutathione conjugation
A primary phase II transformation route is conjugation with glutathione (a tripeptide composed of three amino acids—L-cysteine, L-glutamine, and glycine). Through direct conjugation, it detoxifies many xenobiotics (foreign compounds) and carcinogens, both organic and inorganic. This includes heavy metals, such as mercury, lead, and arsenic. Glutathione conjugation is probably the most important detoxification pathway for industrial toxins and carcinogens and 60% of toxins excreted in the bile are excreted in this way. Glutathione conjugation also produces water-soluble mercaptates which are excreted via the kidneys. The elimination of heavy metals like mercury and lead, is dependent upon adequate levels of glutathione, which in turn is dependent upon adequate levels of methionine and cysteine. When increased levels of toxic compounds are present, more methionine is utilized for cysteine and ultimately glutathione synthesis. Methionine and cysteine have a protective effect on glutathione and prevent depletion during toxic overload. (See the methylation cycle diagram to understand how they are necessary in the creation of glutathione.) This, in turn, protects the liver from the damaging effects of toxic compounds and promotes their elimination.

Glutathione-S-transferase (GST) enzyme family is necessary for glutathione conjugation.

Each subunit of the dimeric GST enzyme has an active site composed of two distinct functional regions: a hydrophilic G-site, which binds the physiological substrate glutathione, and an adjacent hydrophobic H-site for the binding of structurally diverse electrophilic substrates. This structure allows GSTs to catalyze the nucleophilic attack of glutathione on electrophilic substrates, which decreases the electrophile’s reactivity with cellular macromolecules. In humans and rodents, the soluble GSTs are collectively expressed in relatively large amounts, and may comprise as much as 4% of total soluble protein in the liver.

GSTs function by conjugating reduced glutathione (GSH) and catalyzing the attack on foreign compounds or oxidative stress products, generally forming less-reactive materials that can be readily excreted. There are three distinct families of GSTs in mammals: cytosolic, mitochondrial and membrane-associated proteins. Cytosolic GSTs represent the largest family and is subdivided into seven classes based on their amino acid sequence (mu, pi, theta, alpha, sigma, omega and zeta).

Genetics And Glutathione

Polymorphisms are known to occur in the glutathione-S-transferase family and are a significant factor in susceptibility to environmental toxins.

At least five of the human GST genes display functional polymorphisms. These polymorphisms are likely to contribute to the difference between individuals in responses to xenobiotics and clearance of oxidative stress products and have been associated with various diseases.

GST polymorphisms may be significant as studies suggest that that loss of mu, pi and theta GST genes increase susceptibility to inflammatory diseases.

A genetic deficiency of GSTM1 is associated with increased susceptibility to ozone-related asthma as well as susceptibility to aflatoxin B1 (AFB1)-mediated damage to human liver cells. These individuals with GSTM1 polymorphism may benefit from antioxidant supplementation.

The combination of GSTM1 null and GSTP1 Val was significantly associated with an increased risk of lung cancer and hepatocellular carcinoma. An inverse relationship between plasma selenium level, an indicator of the function of glutathione peroxidase and AFB1–albumin adducts were observed to be statistically significant among individuals with null (A null allele is a mutant copy of a gene at a locus that lacks the gene's normal function.) genotypes of GSTM1 and GSTT1, but not among the non-null genotypes. In carriers of hepatitis B virus, there was an increased risk of hepatocellular carcinoma in null genotypes of GSTM1 and T1 but not among individuals who had non-null genotypes. Additionally, GSTM1 null genotype is a risk factor for Alzheimer’s disease and T-2 can suppress drug-metabolizing enzymes such as the GSTs and related oxidative stress-associated pathways

Nuclear factor-erythroid 2-related factor 2 (Nrf2) and GSTs

Nuclear factor-erythroid 2-related factor 2 (Nrf2) transcription controls the formation of glutathione as well as the formation of Glutathione S-transferases (GSTs). Without the glutathione and the glutathione S-transferases there is no glutathione conjugation. Exposure to toxins, such as the mycotoxin called ochratoxin have been shown to inhibit Nrf2 which in turn decreases GSH synthesis. See more about this in the Mycotoxin section below.

 

Inducers, inhibitors and Substrates

Substrate: A substance acted upon by an enzyme such as glutathione-S-transferase. The substrate is the toxin, etc that is to be acted on and undergoes glutathione conjugation.

Inducer: Something that activates glutathione-S-transferases or makes them work better or otherwise induces glutathione conjugation.

Inhibitor: Something that deactivates glutathione-S-transferases or otherwise decreases the enzymes from working as well or otherwise decreases glutathione conjugation.

Substrates and Inducers of Glutathione Conjugation

The reduced glutathione for glutathione conjugation depends on the rate limiting amino acid, cysteine. Cysteine can be made from methionine with the aid of vitamin B6. Additionally, vitamins B2 and B3 are needed for the activity of glutathione reductase, which recycles oxidized glutathione. See more on other necessary nutrients for glutathione here. The basic building blocks of glutathione creation and recycling of glutathione are very important. Without glutathione, there simply will be no glutathione conjugation.

Nrf2 Induction - and therefore induction of glutathione and glutathione S-transferases

The present data indicate that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in regulating mRNA of many phase-II genes including those for glutathione S-transferases. It is also essential for making the substrate, glutathione.

Activators of NRF2 are things like curcumin found in Curcuma longa, silymarin found in Silybum marianum, resveratrol found in Polygonum cuspidatum, as well as grape skin, red wine, and some berries, catechins found in dark chocolate, raw cacao, green tea. One of the strongest activators of NRF2 has been found to be the isothiocyanate called sulforaphane. Sulforaphane is the metabolite of glucoraphanin (GRN) which is contained in the cruciferous plants cell vacuole along with an enzyme called myrosinase (MYR) which are kept separate. When the plant cell wall ruptures and GRN and MYR come together, sulforaphane is enzymatically produced. All the Brassica/Crucifera plant family yields sulforaphane. The highest amounts are in broccoli and to be precise, broccoli sprouts. Cutting, chewing or otherwise opening the cell walls of these plants immediately causes synthesis of sulforaphane. However, it immediately begins to degrade. So, if you make yourself a broccoli drink you need to drink it sooner rather than later.

Johns Hopkins University found that 3-day-old sprouts of cultivars of certain crucifers contained 10–100 times higher concentration of GRN than the corresponding mature plants. Broccoli sprouts also had the added advantage of containing mostly the methylsulfinylalkyl glucosinolate (75% of the total) and very little of the indole glucosinolate found in the mature plant, which is a potential tumor promoter. They theorized that small amounts of broccoli sprouts may protect against cancer as well as larger quantities of the mature vegetable. For more details go here.

D-limonene has been shown to increase total CYP450 activity, and liver GST activity.

Chlorophyllin is a chlorophyll derivative that inhibits CYP450 activity, and stimulates GST activity in cell culture and rodent models. (Additionally, it has been shown to bind toxins.)

Inhibitors of Glutathione Conjugation

Inhibition can take place when there is lack of glutathione production, depletion of glutathione or lack of or depletion of the glutathione-S-transferase enzymes.

When there is more than one compound that is transformed by a single glutathione-S-transferase enzyme, there is what is called competitive inhibition. In this case one compound is unable to be transformed due to the competition of the other compound.

An increase in toxins can also lead to what is called inhibition also due to increased toxic load.

Heavy metals such as mercury, lead and arsenic can deplete glutathione reserves. They lead to oxidative damage by direct generation of free radical species. Lead has been shown to reduce the activity of enzymes that recycle glutathione. Metallothioneins chelate some heavy metals. Metallothioneins have the ability to bind various reactive metal ions including mercury, lead, cadmium, copper, zinc, nickel, cobalt, iron, gold and silver. When there is cellular irritation, especially from oxidative stress, the metallothioneine production is increased. This is stimulated by the activity of Nrf2.

Antimony exposure depletes hepatic glutathione in rats which impairs both glutathione conjugation as well as its function as a general antioxidant.

Glutathione transferases may be inhibited by alcohol, and plant phenols.


Glutathione is also an important antioxidant.

This combination of detoxification and free radical protection, results in glutathione being one of the most important anticarcinogens and antioxidants in our cells, which means that a deficiency is cause of serious liver dysfunction and damage. Exposure to high levels of toxins depletes glutathione faster than it can be produced or absorbed from the diet. This results in increased susceptibility to toxin-induced diseases, such as cancer, especially if phase I detoxification system is highly active, glutathione can't keep up with its antioxidant work and Phase II can't keep up with glutathione conjugation. Disease states due to glutathione deficiency are not uncommon. Health conditions like mold susceptibility (CIRS due to water-damaged buildings), Parkinson's and Alzheimer's have been linked to glutathione deficiency. Glutathione is needed to protect us from damage due to cigarette smoke, radiation exposure, and alcohol to name a few issues. Glutathione provides the major intracellular defense against mercury-induced neurotoxicity. High levels of mercury will use up glutathione.

For details on glutathione, including how to increase glutathione in the body: See more data on Glutathione here.

 

Mycotoxins & Glutathione Conjugation

Some mycotoxins such as aflatoxin are bound to glutathione via conjugation with the assistance of the enzyme glutathione S-transferases. This allows the excretion of mycotoxins out of the body.

Research suggests that mycotoxins can decrease gene expression of the enzymes needed to form glutathione as well as the enzymes for glutathione conjugation. By altering the gene expression for th enzymes needed to make glutathione and for glutathione conjugation, they have now removed one of the major methods our bodies remove mycotoxins and set us up to be unable to remove other enviornmental toxins also. Mycotoxin-related compromise of glutathione production/conjugation can result in an excess of oxidative stress that leads to tissue damage and systemic illness. It can add to the burden and also be causative of the "Chemically Sensitive Person".

Cysteine depletion cause glutathione deficiency. Glutathione may be significantly enhanced and potentially restored by cysteine supplementation. (Cysteine is the rate limiting amino acid necessary to make glutathione.) People who have CIRS due to water-damaged buildings often need cysteine to help make more glutathione. They also need assistance from nrf2 promoters to promote induction of glutathione S-transferases as well as inducing glutathione. See data on this above under Inducers and Subtrates of glutathione conjugation. Also see the Glutathione page for extensive details on substrates and inducers to make glutathione and recycle it.

 

Ochratoxin (OTA) And Glutathion Conjugation

OTA has been shown to increase the formation of oxidative products of lipids, with increased production of malondialdehyde (MDA), a product of the interaction of polyunsaturated lipids and free radicals. It was shown that the toxicity of OTA could be decreased by maintaining glutathione production with N-acetyl cysteine (NAC), which decreased reactive oxygen species (ROS) and 8-oxoguanine formation. These findings suggested that cellular reduced glutathione (GSH) levels play a significant role in limiting the toxicity of the mycotoxin OTA.

OTA appears to inhibit its own detoxification. The tissue injury that is associated with ochratoxin exposure was associated with disruption of pathways related to the transcription factors hepatocyte nuclear factor 4 alpha (HNF4α) and Nrf2. Many Nrf2-regulated genes are involved in chemical detoxification and antioxidant defense. This means, the formation of glutathione and γ-glutamine-cysteine ligase are down-regulated. They are central to the detoxification of OTA. So it appears that OTA decreases formation of the glutathione and related detoxification enzymes for glutathione conjugation necessary for OTA's detoxification. As mentioned above, activators of NRF2 are things like curcumin found in Curcuma longa, silymarin found in Silybum marianum, resveratrol found in Polygonum cuspidatum, as well as grape skin, red wine, and some berries, catechins found in dark chocolate, raw cacao, green tea. One of the strongest activators of NRF2 has been found to be the isothiocyanate called sulforaphane which is found in the Brassicaceae family foods such as broccoli, cauliflower, brussels sprout, and kale.

 

Details for the Chemistry Geeks

Exposure to OTA can down-regulate the formation of glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in GSH synthesis, which will result in a reduction in the intracellular level of GSH. In addition to decreasing GSH, OTA inhibits Nrf2 expression which will decrease the production of the GST isoform GSTP1, which facilitates the detoxification of the lipid peroxidation product 4-hydoxynonenal (4-HNE) by facilitating the conjugation of 4-HNE with GSH. The reactive chemical 4-HNE is known to bind and form adducts to macromolecules including DNA, and cell culture studies demonstrate that Nrf2 and glutathione are needed to protect cells from 4-HNE and lipid peroxidation toxicity.

The findings of increased oxidant stress related to decreased expression of Nrf2 in kidney cells suggests that direct binding of the mycotoxin to DNA is not needed to create DNA damage. The down-regulation of Nrf2 genes results in a decrease in antioxidant defense is due to the decreased expression of Nrf2-related antioxidant genes and thus, the oxidative stress induced by OTA may account for both the genetic damage and the cytotoxicity related to OTA. Thus, the oxidative damage mediated by OTA may be divided into the direct (covalent DNA adduction) and indirect (oxidative DNA damage) mechanisms. Trichothecene type mycotoxins such as T-2 toxin have also been shown to cause cell damage by increasing oxidative stress and depleting glutathione. More can be found on this in this research article.

 

Aflatoxin , Cancer And Glutathione Conjugation

The early observations that an epoxide of aflatoxin B-1 (AFB1) created adducts with DNA led to the concept that mycotoxin-DNA adducts, such as AFB1-DNAs, initiate cancer. This observation has been documented in vitro and in vivo. AFB1 is a known cause of hepatocelllular carcinoma. Some countries such as China and India have great concerns about this mycotoxin contaminating their foods.

Cytochrome P450 enzymes convert aflatoxins to the reactive 8,9-epoxide form (also referred to as aflatoxin-2,3 epoxide in the older literature), which is capable of binding to both DNA and proteins. This CYP450 product can be quite hazardous if not conjugated with glutathione and excreted via the urine. If AFBO is not conjugated and excreted it can cause toxicity including cancer.(Brammler and others 2000) Animal studies have shown AFB1 is primarily conjugated by glutathione conjugation and cytosolic GST activity has been shown to be inversely correlated to the susceptibility of AFB1 carcinogenicity in several animal species. Glucuronidation and sulfation are also used to conjugate AFB1.

Deconjugation can also take place in the gut and cause enterohepatic reciruclation of aflatoxins as well as other conjugated mycotoxins. The intestinal microflora can deconjugate AFB1 and other conjugated mycotoxins and allow them to be reabsorbed.

Aflatoxin and Genetic Diversity/Deficiency

Mechanistically, it is known that the reactive aflatoxin epoxide binds to the N7 position of guanines. Moreover, aflatoxin B1-DNA adducts can result in GC to TA transversions. A reactive glutathione S-transferase system found in the cytosol and microsomes catalyzes the conjugation of activated aflatoxins with reduced glutathione, leading to the excretion of aflatoxin. Variation in the level of the glutathione transferase enzymes as well as variations in the cytochrome P450 system are thought to contribute to the differences observed in interspecies aflatoxin susceptibility.

A genetic deficiency of GSTM1 is associated with susceptibility to AFB1-mediated damage to human liver cells.

Purple Rice Bran Attenuates Aflatoxin B-1 carcinogenicity

Research with purple rice bran extract showed the extract attenuated the aflatoxin B-1 induced initiation state of hepatocarcinogenesis by decreasing Phase I activity and protein expression of CYP1A2, CYP3A and CYP450 reductase, while enhancing the phase II enzymes including GST used for glutathione conjugation and UGT (uridine diphosphate-glucuronosyltransferrases) used for glucuronidation.

Gliotoxin and Patulin

An in vitro study with the mycotoxins gliotoxin and patulin showed a concentration dependent depletion of glutathione when gliotoxin and patulin were added to dendritc cells (antigen-presenting macrophage cells.

Respiratory exposure to gliotoxin or ingestion of patulin increased the asthma-like phenotype in an animal model and additionally increased chronic airway inflammation.

Both gliotoxin and patulin have been shown to shift the Th1/Th2 balance toward a Th2 response.

Citrinin and gliotoxin were shown to decrease glutathione in peripheral blood mononuclear cells and alveolar cells.

A Few General Things to keep in Mind With Mycotoxin Removal

There is little research on the various types of mycotoxins as well as other toxins in water -damaged buildings and how to remove them from the body once a person has inhale, or absorbed them. Most of the research is animal research and surrounds ingestion of mycotoxins. However, this research has a lot of interesting facts that can be gleaned from it and used. Luckily, the interest in this topic is increasing and new research is appearing now. However, we do not have the details that would be helpful im making clear decisions and have to do some guessing and piecing together of data to come up with a game plan cin the clinical setting.

If a person is attempting to remove mycotoxins, but is still living, working, going to school or otherwise spending time in a water-damged building, they will not get well. They must remove themselves from the mycotoxin environment. Later, when their body is able to biotransform better they may be able to handlel these environments better. However, people who are genetically susceptible to issues with water-damaged buildings will do best staying away from them as much as possible.

I often hear people question why it is that a person with "mold sensitivity" or the "chemically sensitive" individual can go to a "clean" environment and heal and then after a period of time (months to years) they can return to past environments and be better able to with-stand the environment that imemediately overwhelmed them previously. In my opinion this is due to the body getting out of the continuous onslaught or toxins long enough to be able to heal the damage from the inflammed gut and brain as well as their neuroendocrine organs healing in general. Additionally, their biotransformational/detox system is allowed to repair and rebuild as needed. This is the short answer to that question as there are many details that are involved in the repair of their body and any organ can be involved in the inflammatory damage, but this is bascially what takes place.

The next question I get in response to the answer I just gave, is "Why can some people with "mold sensitivity, shown by HLA DR haplotypes" clean up their environment or go to a clean environment, get well and then they can spend time in varying degrees of water-damaged buildings without reacting to them?" What it appears to me from my studying this and conversing with people, is as follows: Most people with CIRS due to water-damaged buildings (mold/bacterial/chemical sensitve folks) have had some sort of instigating incident, such as a car wreck, infectious illness, severe emotional upheavel etc that has initiated their problem. Often there was more than one incident all at one time or a series of incidents, each one following the other. This leads to continual inflammation in the body which causes "leaky" gut and blood brain barrier compromise and the biotransformational system of the body can't keep up with what is asked of it. This leads to a down-ward spiral. Once they get out of this spiral and their body repairs itself over a period of time, they now have the fully intact defenses to support and protect them when they return to a less than desireable environment. However, if they are not fully recovered or if they have another instigating incident they can once again be overwhelmed. Each persons genetics play a part in this and this means people have varying degrees of how much of any one toxin or toxins they can withstand. Sometimes they need continual supplements due to some type of inadequacy of their genetics or they may be able to do little about it and just need to stay away from toxins as much as possible. However, most people can get some sort of relief and the more you know about inflammation in the body and support of your biotransformational system, the more you can help your patients.

Bile secretion is critical for removal of conjugated toxins including mycotoxins. Mycotoxins are largely removed through the bile and urine. A person who has light colored stools and may have some difficulty with bacterial gut overgrowth, inability to fully absorb fats ( lighter colored stools, stools tend to float due to fat content, bloating), is a person who may have some issues with flow of bile and further invetigation into this is important. Consider that the bile is released in response to cholecystokinin and deficiency of CCK has been reported in patients with celiac disease, short bowel syndrome, in diabetics, in newborns with infantile colic and in patients receiving total parenteral nutrition. Also consider other causes of decreased bile such as liver and gallbladder inflammation/disease as well as investigate bile acid conjugation, lack of suffiicent cholesterol to make bile acids (Yes, I did say lack of sufficent cholesterol), etc. Some folks with mold sensitivities have lower cholesterol levels in general. They also tend to need some bile stimulation with bitters, or help with bile acid conjugation which is usually helped with taurine. Sometimes both taurine and glycine are helpful. There are many choleretic (bile producing) and cholagogue (bile flow stimulating) herbs that are helpful. A few examples are artichoke, dandelion, and rosemary. As a last resort Ox bile/bile salts are used as a treatment method. Of course it is contraindicated to stimulate bile production if there is any kind of obstructive liver or gall bladder disease. Regarding bile, it is interesting to note that animals tend to loose their appetite when they eat food with mycotoxins. It has been found in research that the peptide hormone cholecystokinin (CCK)is stimulated by the mycotoxins studied. CCK causes increased production of bile and release of bile from the gallbladder as well as digestive enzymes from the pancreas. It additionally decreases appetite and causes nausea. (Another odd thing it does is causes anxiety. - There is an abundance of the peptide in the brain. It is used to cause anxiety in research on drugs to treat anxiety.) So people under constant onslaught from mycotoxins will be releasing CCK more often which will help with bile production but also may produce anxiety. Bile acts as a slight laxative and many people with "mold sensitivity" have looser bowels initially, replaced by constipation sometimes later on. It may be related to excess bile creation and release initially, followed by not enough later in a more chronic stage althugh this is just conjecture on my part.

Heavy metal toxicity can lead to depletion of glutathione and lack of enough glutathione for glutathione conjugation. Heavy metals should be check for. A standard urine test for heavy metals, followed by a provocative urine challange test can give an indication of heavy metals in tissue. Removal of heavy metals is necessary for a person to have a fully functioning biotransformation system including glutathione conjugation ability.

Bile acid binders and mycotoxin binders can be used to remove mycotoxins in food as well as mycotoxins that are being recircualted in the enterohepatic circulation.

Nrf2 activation increases Phase II biortransformation which has been shown to be used for removal of various mycotoxins. Consider using supplements and foods that have been shown to activate Nrf2. Activators of NRF2 are things like curcumin found in Curcuma longa, silymarin found in Silybum marianum, resveratrol found in Polygonum cuspidatum, as well as grape skin, red wine, and some berries, catechins found in dark chocolate, raw cacao, green tea, cinnamaldehyde from cinnamon, caffeic acid in thyme, sage, spearmint, cinnamon, star anise, alpha lipoic acid, alpha tocopherol, lycopene, apple polyphenols (chlorogenic acid and phloridzin), ginkgo biloba, capsaicin found in cayenne, hydroxytyrosol from olives, allyl sulfides from garlic, chlorohyllin in chlorophyll and xanthohumols from hops.

One of the strongest activators of NRF2 has been found to be the isothiocyanate called sulforaphane which is derived from the Brassicaseae family plants such as broccoli as well as plants such as wasabi japonica, and the common radish.

Most people with CIRS due to water-damged buldings (Mold sensitivity is what lots of folks call it) will have excessive inflammation in the body and they initially need to focus on damage caused by it as well as quenching it. Many of the things they would do to decrease the inflammation are supportive of mycotoxins often. You will find some more details about this here.

We need to be more aware of mycotoxins in our food also. People in 1st world countries think their food is safe from mycotoxins but this is not true. There are masked mycotoxins that we don't even test for. Additionally, the change in the worlds climate has caused an increase of mycotoxins on food. Governments make the decision to allow higher levels of mycotoxins in food to keep people from starving and while this is only an issue in third world countries currently, it could easily become an issue everywhere as the climate changes and as we populate the people with an ever increasing amount of humans.

If you found this information helpful, I would appreciate your support in keeping the site going. If you would like to donate to my work, I thank you in advance and send you my deep felt gratitude.

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