The Three Phases of Detoxification
Phase I Detoxification: Enzymatic Transformation
Under most circumstances, phase I enzymes begin the detoxification process by chemically transforming lipid soluble compounds into water soluble compounds in preparation for phase II detoxification. The bulk of the phase I transformation reactions are performed by a family of enzymes called the cytochrome P450s (CYPs).
CYP enzymes are relatively non-specific, each has the potential to recognize and modify countless different toxins; after all, a mere 57 human CYPs must be able to detoxify any potential toxin that enters the body.20 However, the cost of this versatility is speed; CYPs metabolize toxins very slowly compared to other enzymes. For instance, compare the predominant CYP3A4, which metabolizes 1‒20 molecules per second,21 to superoxide dismutase (SOD), which metabolizes over a million molecules per second. Major sites of detoxification overcome the slower speed by producing large amounts of CYPs; CYPs may represent up to 5% of total liver proteins, and similar large concentrations can be found in the intestines. CYPs are amongst the most well studied and best characterized detoxification proteins due to their role in the metabolism of prescription drugs, and to their role in metabolizing endogenous biochemicals (for example, aromatase, which transforms testosterone to estradiol, is a CYP).22
Several other enzymes contribute to the phase I process as well, notably the flavin monooxygenases (FMOs; responsible for the detoxification of nicotine from cigarette smoke); alcohol and aldehyde dehydrogenases (which metabolize drinking alcohol); and monoamine oxidases (MAO’s; which break down serotonin, dopamine, and epinephrine in neurons and are targets of several older antidepressant drugs).23
Phase II Detoxification: Enzymatic Conjugation
Following phase I transformation, the original lipid-soluble toxin has been converted into a more water-soluble form. However, this reactive intermediate is still unsuitable for immediate elimination from the cell for a couple of reasons: 1) phase I reactions are not sufficient to make the toxin water-soluble enough to complete the entire excretion pathway; and 2) in many cases, products from the phase I reactions have been rendered more reactive then the original toxins, which makes them potentially more destructive than they once were. Both of these shortcomings are addressed by the activities of the phase II enzymes, which modify phase I products to both increase their solubility and reduce their toxicity. The activation of the phase II enzymes is responsible for the anti-mutagenic and anti-carcinogenic properties of the metabolic detoxification systems; it is widely accepted that phase II enzymes protect against chemical carcinogenesis, especially during the initiation phase of cancers.24
At the genetic level, the production of most phase II enzymes is controlled by a protein called nuclear factor erythroid-derived 2 (Nrf2), a master regulator of antioxidant response.25 Under normal cellular conditions, Nrf2 resides in the cytoplasm (the liquid inside cells within which the cells components are contained) of the cell in an inactive state.26 However, the presence of oxidative stress (triggered by metabolism of toxins by CYPs) activates Nrf2, allowing it to travel to the cell nucleus.27 In the cell nucleus, Nrf2 turns on the genes of many antioxidant proteins, including the phase II enzymes.28 In this way, Nrf2 “senses” oxidative stress or the presence of toxins in the cell, and allows the cell to mount an appropriate response. Nrf2 regulates the activity of genes involved in the synthesis and activation of important detoxification molecules including glutathione and superoxide dismutase (SOD). It also plays an important role in initiating heavy metal detoxification, and the recycling of CoQ10, a potent antioxidant.29-31
Certain dietary constituents (including sulforaphane from broccoli and xanthohumol from hops) may also directly activate Nrf2 and stimulate antioxidant enzyme activity; this may partially explain their beneficial effects on detoxification.32
There are several families of phase II enzymes that differ significantly in their activities and biochemistry. In several cases, phase II enzymes exhibit redundancy—a particular xenobiotic or endobiotic can be detoxified by more than one phase II enzyme.
UDP-glucuronosyltransferases. UDP-glucuronosyltransferases (UGTs) catalyze glucuronidation reactions, the attachment of glucuronic acid to toxins to render them less reactive and more water-soluble. There are several different UGTs that are distributed throughout the body, with the liver being the major location. In humans, many xenobiotics, environmental toxicants, and 40‒70% of clinical drugs are metabolized by UGTs.33 The plasticizer bisphenol A34 and benzopyrene (from cooked meats)35 are two notable examples of UGT substrates (a substrate is a molecule upon which an enzyme acts). Intestinal UGTs may affect oral bioavailability of several drugs and dietary supplements, and may be responsible for chemoprevention in this tissue.36
Glutathione S-transferases. Glutathione S-transferases (GSTs) catalyze the transfer of glutathione (a significant cellular antioxidant) to phase I products. GSTs play a major role in the metabolism of several endobiotics, including steroids, thyroid hormone, fat-soluble vitamins, bile acids, bilirubin and prostaglandins.37 GSTs can also function as antioxidant enzymes, detoxifying free radicals38 and oxidized lipids or DNA.39 GSTs are soluble enzymes that are ubiquitous in nature and in humans, forming about 4% of the soluble protein in the human liver and present in several other tissues (including brain, heart, lung, intestines, kidney, pancreas, lens, skeletal muscle, prostate, spleen and testes).40,41 Products of GST conjugation can be excreted via bile, or can travel to the kidneys where they are further processed and eliminated in urine.
Sulfotransferases. Sulfotransferases (SULTs) attach sulfates from a sulfur donor to endo- or xenobiotic acceptor molecules. This reaction is important both in detoxification reactions, as well as normal biosynthesis (the addition of sulfate to chondroitin and heparin, for example, is catalyzed by specific SULTs).42 SULTs play a major role in drug and xenobiotic detoxification, and the metabolism of several endogenous molecules (including steroids, thyroid and adrenal hormones, serotonin, retinol, ascorbate and vitamin D).43 SULTs in the placenta, uterus, and prostate are thought to play a role in the regulation of androgen levels.44 In contrast to other phase II enzymes, SULTs can convert a number of procarcinogens (such as heterocyclic amines from cooked meats) into highly reactive intermediates which may act as chemical carcinogens and mutagens.45
While the UGTs, GSTs, and SULTs catalyze the bulk of human detoxification reactions, several other phase II enzymes contribute to the process to a lesser, but still important extent, including:
- Methyltransferase enzymes. Methyltransferase enzymes catalyze methylation reactions using S-adenosyl-L-methionine (SAMe) as a substrate. Catechol O-methyltransferase (COMT) is a major pathway for eliminating excess catecholamine neurotransmitters (such as adrenaline or dopamine). Methylation reactions are one of the few phase II reactions that decrease water solubility46;
- Arylamine N-acetyltransferases (NATs). NATs detoxify carcinogenic aromatic amines and heterocyclic amines47;
- Amino acid conjugating enzymes. Acyl-CoA synthetase and acyl-CoA amino acid N-acyltransferases attach amino acids (most commonly glycine or glutamine) to xenobiotics. The food preservative benzoic acid is one example of a toxin metabolized by amino acid conjugation.48
Phase III Detoxification: Transport
Phase III transporters are present in many tissues, including the liver, intestines, kidneys, and brain, where they can provide a barrier against xenobiotic entry, or a mechanism for actively moving xenobiotics and endobiotics in and out of cells.49 Since water-soluble compounds require specific transporters to move in and out of cells, phase III transporters are necessary to excrete the newly formed phase II products out of the cell. Phase III transporters belong to a family of proteins called the ABC transporters (for ATP-binding cassette),50 because they require chemical energy, in the form of ATP, to actively pump toxins through the cell membrane and out of the cell.51 They are sometimes called the multidrug resistance proteins (MRPs), because drug-resistant cancer cells use them as protection against chemotherapy drugs.52
In the liver, phase III transporters move glutathione, sulfate, and glucuronide conjugates out of cells into the bile for elimination. In the kidney and intestine, phase III transporters can remove xenobiotics from the blood for excretion from the body.53