Phase II might just be the most important step of your body’s amazing detox system. Find out why and how to support this in this post.
Phase II Detoxification
Recap: Detox Phases
Our bodies process and remove these foreign chemicals (xenobiotics) thanks to our efficient detoxification mechanisms. These mechanisms also deal with metabolic products such as excess hormones (endobiotics).
“Enzymes of detoxification” are a big family of enzymes that alter foreign chemicals and metabolic waste either by making them more readily excretable or less pharmacologically active [1].
The detoxification process can be roughly divided into three phases [2]:
- Phase I is governed by transformation enzymes – these enzymes oxidize, reduce and hydrolyze toxins/drugs
- Phase II is managed by conjugation enzymes – these enzymes conjugate Phase I products
- Phase III is carried out by transport proteins – these proteins transport the final products from the cell
Phases I, II, and III must work in unison for the proper removal of unwanted toxins, drugs, and excess hormones.
Enzymatic Conjugation Reduces the Toxicity of Phase I Products
Through phase I transformation, the fat-soluble toxin is converted into a more water-soluble form. However, phase I reactions are not always sufficient to make the toxin water-soluble enough and in many cases, they render the products more reactive, which makes the toxins potentially more destructive. This is where the phase II enzymes kick in [3].
Phase II enzymes increase the solubility and reduce the toxicity of phase I products [3].
Scientists think they play a major role in the cellular detoxification of damaging, genotoxic and carcinogenic chemicals [4].
Gene polymorphisms and/or a lack of these enzymes have been suggested to play a role in several forms of cancer, though far more research is needed to verify this link [4].
Phase II Enzymes
Phase II enzymes contain several superfamilies of conjugating enzymes. Among the most important ones are [5]:
- UDP-glucuronosyltransferases (UGT)
- Glutathione S-transferases (GST)
- Sulfotransferases (SULT)
- N-acetyltransferases (NAT), and
- Methyltransferases (MT).
UDP-glucuronosyltransferases (UGTs)
UDP-glucuronosyltransferases (UGTs) are responsible for the glucuronidation reaction, one of the most important detoxification pathways of the Phase II transformation [6].
UGT enzymes are responsible for the metabolism of many xenobiotics (drugs, chemical carcinogens, environmental pollutants, and dietary substances) and endobiotics (bilirubin, steroid hormones, thyroid hormones, bile acids, and fat-soluble vitamins) [3].
In humans, approximately 40-70% of all clinical drugs are metabolized by UGTs [3].
Monoamine neurotransmitters, dopamine, and serotonin are also processed by UGTs [6].
UGTs are found throughout the body, in the intestines, kidney, brain, pancreas, and placenta, but most of them are found in the liver [3].
In the brain, UGTs actively participate in the overall protection against the intrusion of potentially harmful substances [6].
There are 18 functional UGT enzymes in humans, belonging to two gene families, UGT1 and UGT2, that are, further divided into three subfamilies: UGT1A, UGT2A, and UGT2B [7].
Some studies suggest that UGTs process and inactivate toxins such as:
- Cancer-causing polycyclic aromatic hydrocarbons (PAHs) [8], including benzopyrene, found in cigarette smoke, wood smoke, and burnt foods [9].
- Bisphenol-A (BPA), a ubiquitous environmental toxin used in plastics and associated with various human diseases including breast cancer in limited studies [10].
- Some nitrosamines found in tobacco smoke [11].
- Heterocyclic amines (HACs), found in red processed meat. 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), the most mass abundant carcinogenic HA found in well-done cooked meat, is extensively glucuronidated by UGT1A proteins [12].
- Some fungal toxins found in crops [13].
- Aromatic amines [14].
UGTs also remove “used” hormones, such as estrogen, T3, and T4, that are produced naturally by the body [15, 16].
UGT1A1 is the only UGT responsible for bilirubin glucuronidation [3].
UGT2B7 is the major enzyme responsible for the glucuronidation of opioids [3].
According to some estimates, over 10% of the population have hereditary deficiencies in UGTs [17]. Hence, UGTs have been hypothesized to constitute an important determinant of susceptibility to chemical carcinogenesis, teratogenesis, and neurodegeneration [17].
Enhanced UGT activities might lead to more efficient detoxification of carcinogenic compounds. Some scientists believe they might contribute to the prevention of gastrointestinal and other cancers, but this hasn’t been confirmed in humans [18].
We differ in UGTs
Patients with a very rare inherited disorder, the Crigler-Najjar syndrome have a complete or partial absence of UGT1A1. Complete UGT1A1 absence results in high levels of unconjugated bilirubin, severe jaundice, and often brain damage in infants. Patients with a partial deficiency of this enzyme are less severely jaundiced and generally survive into adulthood without neurological or intellectual impairment [3].
Gilbert syndrome is an autosomal dominant hereditary disorder caused by a mutation in the UGT1A1 gene causing mild hyperbilirubinemia. Gilbert syndrome is found in approximately 10% of the population [3] and has been strongly associated with altered xenobiotic glucuronidation [19].
According to another study, a UGT1A1*28 gene variant results in 30 – 40% lower UGT1A1 levels. When people with this variant are exposed to well-cooked red meat, they exhibit higher urinary mutagenicity (in other words, less of the mutagenic compounds are inactivated) [20].
Factors that Influence Glucuronidation (UGTs)
- Analgetics, nonsteroidal anti-inflammatory drugs (NSAID), antiviral drugs, anticonvulsants, and anxiolytics/sedatives can inhibit drug glucuronidation [3].
- Further, some drugs (analgesics, antivirals, and anticonvulsants) may also act as UGT- inducers [3].
- Cruciferous vegetables, resveratrol, and citrus seem to induce UGT enzymes. However, the effects vary depending on gender and gene variants [21].
- Dandelion, rooibos tea, honeybush tea, rosemary, soy, ellagic acid, ferulic acid, curcumin, and astaxanthin potentially enhanced UGT activity in animal studies [21].
- Lycopene, a red pigment found in tomatoes, carrots, and watermelon, increases UGT activity [22].
Beta-glucuronidase
Beta-glucuronidase is an enzyme produced by gut bacteria and intestinal cells. Beta-glucuronidase removes (deconjugates) glucuronic acid from neutralized toxins – reversing the reaction catalyzed by UGTs, and reverting the toxins to their previous form [21].
A high-fat diet may increase beta-glucuronidase activity, according to animal experiments [21].
Elevated beta-glucuronidase activity has been associated with an increased risk of various cancers, particularly hormone-dependent cancers such as breast, prostate, and colon cancers in limited studies. More research is needed [23].
Polyphenol extracts of certain berries, specifically strawberries and blackcurrant inhibit beta-glucuronidase activity in animal studies. Human data are lacking [21].
D-glucaric acid, found in many fruits, vegetables, and legumes, may inhibit this enzyme, but human studies have failed to prove this [21].
Glutathione S-transferases (GSTs)
Glutathione S-transferases (GSTs) catalyze the transfer of glutathione – an important cellular antioxidant, to xenobiotic compounds [24].
Apart from processing xenobiotics, GSTs protect against oxidative stress [3], and function as a defense against reactive oxygen species (superoxide radical and hydrogen peroxide) that arise through normal metabolic processes. Many of these are formed by phase I reactions catalyzed by CYPs and other oxidases [3].
These enzymes also participate in the binding and transport of bilirubin, prostaglandins, and glucocorticoid, thyroid, and steroid hormones [25, 26, 3].
GSTs are widely distributed throughout the body and can be found in liver, kidney, brain, pancreas, testis, heart, lung, small intestine, skeletal muscle, prostate and spleen [3].
There are many GSTs, divided into two distinct super-families: the membrane-bound microsomal and cytosolic GSTs. Microsomal GSTs play a key role in the endogenous metabolism of leukotrienes and prostaglandins. Human cytosolic GSTs are highly polymorphic and can be divided into six classes: α, µ, ω, π, and ζ (designated by Greek letters) [27].
Scientists think that GSTs process and inactivates toxins such as:
- α,β-unsaturated carbonyls, many of which are toxic, mutagenic and carcinogenic [3].
- Polycyclic aromatic hydrocarbons (PAHs), which are found in cigarette smoke, diesel fuel and grilled meats [26].
Extended life span in animals was associated with significantly higher levels of GSTs, but we can’t draw any longevity-related conclusions from studies on fruit flies. Whether or not GSTs play a role in human lifespan is still unknown [28].
We differ in GSTs
Depending on the population, approximately 50% of people lack GSTM1 and the same is true for GSTT1. GSTM1 deletion was seen in 53% of whites, in 40 – 60% of Asians, and 21% of African Americans, while GSTT1 deletion was found in 18% of whites, 22% of African Americans, and 45 – 60% of Asians [24, 29].
The dual GSTM1/GSTT1 deletion was associated with higher serum iron and total and LDL-cholesterol concentrations, and lower malondialdehyde concentrations [24].
Lack of GSTM1, GSTT1, and GSTP1 genes was associated with a higher incidence of some cancers, but the associations were weak. Loss of these genes has also been found to be somewhat associated with increased susceptibility to asthma and allergies, atherosclerosis and rheumatoid arthritis. More research is needed [3].
Limited studies have associated variants in omega-class GSTs with the age of onset of neurological diseases such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis. Further human studies are required to verify this link [30].
Factors that May Influence GSTs
- Synthetic and naturally occurring phenols, quinones, dopamine or derivatives of vitamin C inhibit GSTs [3].
- Extracts of Ginkgo biloba have been found to induce GSTP1 and elevated cellular GST activity [3].
- Extracts from cruciferous vegetables (e.g. broccoli, Brussels sprouts, cabbage) as well as grapefruit extract, and limonene (found in citruses) induce GSTs [3, 31, 32].
- Allium vegetables, resveratrol, fish oil, black soybean, purple sweet potato, curcumin, green tea, rooibos tea, honeybush tea, ellagic acid, rosemary, ghee, and genistein also induce GSTs [21, 32].
- GSTs are also induced by butyrate, a product of gut flora-derived fermentation of plant foods [33].
- Insulin administration increases GST, while glucagon decreases GST gene expression [34].
However, many of these interactions were only studies in animals or cells. More human data are needed.
Nutrients that may increase the production of glutathione, essential for GST function include vitamin B6, magnesium, selenium, curcuminoids (from turmeric), silymarin (from milk thistle), folic acid, alpha-lipoic acid, cruciferous vegetables and artichoke [21].
Sulfotransferases (SULTs)
Sulfotransferases (SULTs) are responsible for sulfonation, a reaction that attaches sulfates to endo- or xenobiotics.
SULTs are important for the biotransformation of numerous xenobiotics such as drugs and chemicals [3], but they also play a significant role in the metabolism of endogenous compounds, such as steroids, catecholamines, serotonin, iodothyronines, eicosanoids, some tyrosine-containing peptides, retinol, 6-hydroxymelatonin, ascorbate and vitamin D [3].
On the other hand, a number of compounds (procarcinogens) can be converted by SULTs into highly reactive intermediates which can act as chemical carcinogens and mutagens by damaging DNA [3].
In humans, three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members [35].
Researchers suggest that SULTs process and inactivate toxins such as:
- monocyclic phenols [3].
- naphtols [3].
- benzylic alcohols [3].
- aromatic amines [3].
- hydroxylamines (possible mutagens) [3].
SULTs also process and inactivate dopamine and iodothyronines [3].
Sulfonation of aromatic hydroxylamines by SULT1A2 is an example of a toxification reaction, where the sulfoconjugates of hydroxylamines are chemically reactive and mutagenic, and therefore more toxic than the original compounds [3].
We differ in SULTs
A mutation in SULT1A1, found in 25.4 – 36.5% of Caucasians, leads to reduced sulfotransferase activity [3].
Factors that May Influence SULTs
- Grapefruit juice, orange juice, green tea, black tea, and oolong tea can inhibit SULTs [3].
- Curcumin is a potent inhibitor of SULT1A1 in the human liver [3].
- Quercetin inhibits SULT1A1 [3].
- Some non-steroidal anti-inflammatory agents can inhibit SULT1A1 and SULT1E1 [3].
- Wine anthocyanins and flavonols, apple and grape juice, catechins, quercetin, curcumin, resveratrol, flavonoids (apigenin, chrysin, fisetin, galangin, kaempferol, quercetin, myricetin, naringenin, and naringin), and certain phytoestrogens (daidzein, genistein) may also inhibit SULTs, but these have yet to be proven in human and animal studies [21].
- Retinoic acid can increase SULTs [3].
- Genistein, a natural isoflavone found in soybean products, induces SULT1A1 and SULT2A1 [3].
The extent of and relevance of these interactions in humans still remains uninvestigated.
N-acetyltransferases (NATs)
Arylamine N-acetyltransferases (NATs) are responsible for acetylation – a major route of biotransformation for many arylamine and hydrazine drugs, and carcinogens present in the diet, cigarette smoke and the environment [36].
The role of NATs in endogenous metabolism is unclear, but it may be linked with folate metabolism [3, 37].
There are two NAT enzymes: NAT1 and NAT2 [36].
NATs have been suggested to process and inactivate toxins such as:
- aromatic amines, many of them carcinogens [3].
- hydrazine, a highly toxic foaming agent used for industrial and pharmacological purposes [3].
- drugs such as isoniazid (antituberculosis drug), hydralazine (antihypertensive drug) and sulphonamides (antibacterial drugs) [3].
We differ in NATs
Based on the differences in NAT genes, humans can be slow, intermediate or rapid acetylators [3].
When it comes to NAT1, about 8% of the individuals are slow acetylators, but the percentage can be much higher in some regions of the world, such as Lebanon [36].
In Caucasian and African populations, the frequency of the slow NAT2 acetylation phenotype varies between 40 and 70%, while that of Asian populations, such as Japanese, Chinese, Korean, and Thai, ranging from 10 to 30% [36].
Rapid NAT2 acetylator phenotype has been linked to the increased risk associated with Alzheimer’s disease. By contrast, the slow phenotype appears to increase the risk of Parkinson’s disease. These associations haven’t been verified in large-scale studies [36].
In other studies, the rapid acetylator phenotype emerged as a potentially strong risk factor for colorectal cancer in those individuals who have a higher exposure to food-derived heterocyclic amines [36]. But there was also an association between NAT2 slow acetylation genotype and the risk of developing several other forms of cancer, though more research is needed [3].
An interaction between NAT1 polymorphism, lack of maternal multivitamin use and association with birth defects have been suggested [3].
Factors that May Influence NATs
- Caffeic acid, esculetin, quercetin, kaempferol, and genistein inhibit NAT1 [3].
- Scopoletin and coumarin inhibit NAT2 [3].
- Diallyl sulfide (DAS) and diallyl disulfide (DADS), major components of garlic, inhibit NAT activity [3].
- Androgens induce NAT1 [3].
The impact of these factors on NAT1 and health in humans is still unknown.
Methyltransferases TPMT and COMT
There are many different methyltransferases. TPMT and COMT are two important methyltransferases involved in detoxification.
TPMT
Thiopurine S-methyltransferase (TPMT) catalyzes the methylation of aromatic heterocyclic sulfhydryl compounds including anticancer and immunosuppressive thiopurines (drugs used to treat acute lymphoblastic leukemia, autoimmune disorders, inflammatory bowel disease, and organ transplant recipients) [3].
Impaired activity of TPMT causes an accumulation of thiopurine nucleotides, which can lead to cytotoxicity and to the failure of blood cell production (hematopoiesis) [3].
TPMT levels are highest in the liver and kidney and relatively lower in the brain and lungs [3].
No endogenous substrate is known for this enzyme and its biological role remains unidentified.
TPMT genetic variants
Approximately 1 in 300 Caucasians are homozygous for a defective allele or alleles for the trait of very low activity, ∼11% of people are heterozygous and have intermediate TPMT activity [3].
Humans with genetically determined low or intermediate TPMT activity seem to have a higher risk for side-effects when treated with standard doses of thiopurines. On the other hand, individuals with high TPMT activity have a lower risk of toxicity but the optimal concentration of drugs in the blood cannot be achieved. In this instance, an increased risk of leukemia relapse has been suggested, but more research is needed [3].
COMT
Catechol-O-methyltransferase (COMT) is responsible for methylation of the catecholamine transmitters (norepinephrine, epinephrine, and dopamine). COMT plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function and pain processing. COMT also processes drugs with catechol structure used in the treatment of hypertension, asthma and Parkinson’s disease [3].
COMT genetic variants
The level of COMT enzyme activity (low, intermediate and high levels) is genetically polymorphic. A common functional genetic polymorphism in the COMT gene may contribute to the etiology of alcoholism [3].
Factors that May Influence TPMT and COMT
- TPMT activity is higher in men than women [3].
- Naproxen, mefenamic and tolfenamic acid, Olsalazine, 5-aminosalicylic acid and sulphasalazine may inhibit TPMT [3].
- Ketoprofen and ibuprofen are weak inhibitors of TPMT [3].
- Flavonoid quercetin found in green tea may inhibit COMT [3].
- A high sucrose diet may inhibit COMT [21].
These nutrient cofactors and methyl donors support the methylation process: methionine, vitamin B12, vitamin B6, betaine, folate, and magnesium [21].
Human data are largely lacking.
Amino acid conjugating enzymes
Amino acid conjugation is a detoxification pathway for a limited number of xenobiotic carboxylic acids. It involves two groups of enzymes, Acyl-CoA synthetases, and Acyl-CoA amino acid N-acyltransferases [38].
Not much is known about their detoxification roles yet [38].
Glycine conjugation prevents the accumulation of benzoate, a commonly used preservative [39].
Why the Phase II/Phase I Activity Ratio is Important
Phase I detoxification can produce toxic metabolites. This is not a problem when the phase II enzymes are working well because they efficiently neutralize toxic phase I products as they form.
However, studies suggest that this balance between phase I and phase II activity can become uncoupled in some cases, leading to the production of toxic metabolites that cannot be timely detoxified, potentially causing cellular and DNA damage [40].
Factors that May increase phase I over phase II metabolism
These detrimental factors include:
- smoking – PAHs from cigarette smoke [40].
- aryl amines from charbroiled meats [40].
- alcohol consumption [40].
Glucocorticoid and anticonvulsant drugs may also increase phase I over phase II enzymes, but these drugs have legitimate medical indications (such as inflammatory diseases and epilepsy) [40].
Factors that May Increase Phase II activity by activating Nrf2
Factors that may activate Nrf2 might also hypothetically increase phase II activity. However, have in mind that this is largely theoretical and human data to back up this approach are lacking.
Nrf2 (Nuclear factor erythroid 2 related factor 2) is a major regulator of Phase II enzymes [2].
Nrf2 is activated by oxidative stress [41]. Several cancer preventive agents, GSH-depleting agents, electrophiles, and heavy metals are also known to induce the expression of Nrf2 [42].
Nrf2 activators can control or prevent a wide array of human diseases associated with oxidative stress [42].
A number of plant-derived compounds with antioxidant activities have been hypothesized to accomplish their beneficial effects through Nrf2 and subsequent Phase II detoxification:
- Isothiocyanates, such as sulforaphane found in broccoli [42, 43], and 6-methylsulfinylhexyl isothiocyanate (6-HITC) found in Japanese horseradish [44, 45].
- Resveratrol, found in peanuts, grapes, and red wines [42].
- Ellagic acid found in blackberries, cranberries, pecans, pomegranates, raspberries, strawberries, walnuts, wolfberries, and grapes [40, 46].
- Curcumin found in turmeric [42].
- CAPE, caffeic acid phenethyl ester; from honeybee hives [42].
- Epigallocatechin gallate, from green tea [42, 47].
- Allyl sulfides found in garlic [42].
- Xanthohumol, a compound found in hops [42, 48].
- Cinnamaldehyde found in cinnamon [42].
- Hydroxytyrosol from olives [49].
- Capsaicin, found in chili peppers [50].
- Apple polyphenols [51].
Some scientists think that the enhancement of Phase II activity can explain, at least in part, the health-boosting effect of fruits and vegetables, and their cancer-preventing properties. Human studies are needed to support their claims, though [40].
These supplements have also been suggested to increase Nrf2 activity in animals and cells:
- Alpha-tocopherol (vitamin E) [52]
- Chlorophyllin [53]
- Alpha-lipoic acid [54]
- Dithiolethiones such as Oltipraz, an organosulfur cancer-preventive compound [42], and flavonoids such as Chalcone [55].
Human data are lacking.
