|Phase I and Cytochrome P450 Pathway|
This page contains educational material about the Cytochrome P450 detoxification pathway of the body. 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.
Phase I Transformation (Detoxification) Explained
For preliminary information introducing you to what biotransformation/detoxification is all about, go to this biotransformation/detoxification page.
The body has provided an enzymatic biotransformational/detoxification system to change lipid-soluble or non-polar, non water soluble toxins into less active/less toxic water-soluble substances. Generally this is done via the enterplay of Phase I and Phase II enzymatic system. In this section, we will look at the Phase I enzymatic system.
In phase 1, enzymes act on a toxic or reactive compound to transform the compound into a form that is more water soluble and can be excreted or now has sites that can be used as conjugation (attachment) sites by Phase II. These sites are created by either uncovering a reactive group or adding a reactive group to the toxin. Generally, the idea is for phase I to make the substances less toxic, but sometimes the transformation makes them more toxic. This is called a bioactivation reaction. When this happens, if the new, more reactive substance is not immediately conjugated by Phase II, it can cause more damage than the original substance would have. This is an issue if Phase II is not working up to par for one of various reasons.
Phase I is our first defense against toxins. Usually, oxidation (oxidative reaction), reduction (reductive reaction) and/or hydrolysis (hydrolytic reaction) reactions are used to expose or add a functional group. Dehalogenation may also be used. Depending on the type of molecule being dealt with, this is usually either a hydroxyl , a carboxyl, or an amino group that is exposed. Usually, the compound will then be ready to undergo Phase II at this point. Sometimes, the new compound will be ready to eliminate immediately after Phase I.
Oxidation is the most common route of transformation of xenobiotics. In oxidation you add an oxygen and remove a hydrogen. This happens with oxidases, alchohol dehydrogenases, and cyp450 enzymes. In reduction you remove oxygen and add a hydrogen with the help of reductase enzymes. In hydrolysis you add water with the help of esterases, phosphatases and others. Here are some slides if you are interested in learning more.
Main Enzymes Involved in the Oxidative Reactions
Aldehyde oxidase (AO)
Cytochrome P450s (P450s)
Flavin-containing monooxygenases (FMOs)
Monoamine oxidases (MAOs)
Xantine oxidase (XO)
Main Enzymes Involved in the Hydrolytic Reactions
others: choiniestereases, paraoxonases
Phase I transformation of toxins involves a large group of isoenzymes. Although there are several types of phase I enzymes, the most common enzymes are collectively called the cytochrome P450 (CYP450) system. (The P stands fro pigment and the 450nm is the wavelength of light absorption.) The CYP450 system is also known as the NADPH-CYP450 system because it uses oxygen and the co-factor NADPH. CYP contains an iron protoporphyrin prosthetic group (heme). CYP450 catalyzes primarily oxidation, reduction, and hydrolysis, reactions which generally expose a functional group. This is followed by the Phase II processes of thiol conjugation, glucuronidation, glutathione conjugation, amino acid conjugation, sulfation or acetylation which adds a compound to the functional group exposed in Phase I. Ultimately the process is changing a lipophilic or fat loving compound into a more polar or water loving compound which is now in a form easier for the body to remove. The classic reaction is: NADPH + H + O2 +RH ----> NADP+ H2O +R-OH (RH is the toxin)
Among phase I enzymes, the CYP450 superfamily plays a key role in the metabolism of both endogenous and exogenous molecules, such as steroids, fatty acids, carcinogens, toxins and 90% of drugs currently in use. It should also be mentioned that the CYP450 system is not just removing toxins, it is also producing some compounds such as cholesterol, steroids, fatty acids and vitamin D.
In humans more than 50 enzymes make up the cytochrome P450 system. It is thought humans developed so many isoenzymes to keep up with the new alkaloids made by plants. The idea is that humans and other animals developed new isoenzymes to metabolize new plant alkaloids, the plants then developed new alkaloids and the animals respond again etc. It is known that the gene for CYP450 has existed for more then 3.5 billion years. Each enzyme works best in detoxifying certain types of chemicals, but with considerable overlap in activity among the enzymes, the system allows for some degree of back up assistance if an enzyme is not available. Each enzyme is encoded by separate genes. Most CYP450 genes are subject to genetic polymorphism which means we all have different abilities to transform toxins.
The CYP450 enzymes are categorized into families and subfamilies. They are classified into different families according to the amino acid similarities of the encoding proteins. CYP450 genes in families 1–3 encode for 22 different isoforms, mainly involved in the metabolism of drugs and other xenobiotics, whereas families 4–51 are generally involved in the biosynthesis and metabolism of endogenous compounds.
The CYP450 are heme based enzymes located in the smooth endoplasmic reticulum, largely concentrated in liver cells and mucosal intestinal cells but also found in a variety of other bodily systems to a lesser degree. The brain has additionally been identified as having a lot of P450 enzyme activity. The primary role for the P450 system seems to be one of metabolism and detoxification of endogenous compounds after they have been consumed as part of food products. This accounts for the high concentrations of these enzymes located in the liver and small intestine. As much as 5% of liver proteins have been found to be CYP450 enzymes.
When the metabolites of Phase I are made in excess of what Phase II can conjugate, there is a back up of toxic metabolites and this can be a problem. Phase II must be competent and able to handle Phase I metabolites to protect the body against diseases such as cancer.
Antioxidants needed for protection from free radicals
So, you now know that stimulation of phase I is contraindicated if the patient’s phase II systems are underactive. Additionally, you have to be aware that increasing or decreasing Phase I will alter how many drugs are metabolized and may cause trouble for people who are on medications.
Several CYP enzymes have been identified as more important than others in oxidative metabolism. They are CYP3A4 ( the most important), CYP2D6, CYP2C9, and CYP2C19. Other notable CYPs are CYP2E1, CYP2A6 and CYP1A2.
Inducers, inhibitors and Substrates
You will see people talk about substrates of CYP450 system and Inducers of CYP450. Both will increase its activity. However, be aware that substrates are necessary items for the system to work efficiently, while inducers are anything that will cause its activity. A vitamin may be a substrate which will help induce CYP450, while coffee would be an inducer and not a substrate. Some foods and herbs are inducers. Some of the research on inducers and inhibitors of CYP450 is done in such a way that we can not really know for sure what is happening in the body. This is especially true when only a constituent of a food is studied. We can't assume the whole food will have the same effect. One food or herb can have both inducers and inhibitors in the item. Still it is good to know about this data. Some foods such as grapefruit have definitely been shown clinically to be inhibitors of CYP450.
A considerable amount of research has found that various substances activate cytochrome P450 enzymes while others inhibit it.
Obviously, it is beneficial to improve phase I transformation in order to eliminate toxins as soon as possible. This is best accomplished by providing the needed nutrients and non-toxic stimulants while avoiding those substances that are toxic.
Enzyme induction occurs for CYP450 with appropriate substrates (such as vitamins and minerals). The activity of CYP450 oxidases vary across the population due to polymorphism from one person to another. This is seen clinically in the different reactions people have to drugs or especially when multiple drugs are co-administered. Most serious drug interactions are due to the interference of the metabolic clearance of one drug by another drug. One drug causes an increase or decrease in the CYP450 enzyme activity. This then causes an increase or decrease in how fast the body removes the other drug from the body. This can cause either an underdosage or overdosage of a drug. In some cases this leads to serious clinical consequences, including death. It is not just drugs that effect the CYP clearance of other dugs. Foods, herbs, and supplements can effect the CYP enzymes and can cause mild to significant changes in how the CYP enzymes are working.
When there is a high load of toxins, the body can respond by increasing the necessary enzymes. A toxin that upregulates enzymes can selectively upregulate just one enzyme, or it may upregulate many enzymes.
Substrates that are needed for CP450 system
Deficiencies in vitamins A, B2 and B3, folate, C, E, iron, calcium, copper, zinc, magnesium, selenium have all been shown to decrease the activities of one or more phase I enzymes, or slow the transformation of specific drugs. So, it can be assumed that all of these nutrients are probably necessary for induction of Phase I.
Brassica family such as cabbage, broccoli, and brussels sprouts; charcoal-broiled meats; high-protein diet; Limonene in oranges and tangerines (but not grapefruits)
Specific Information on Inducers of phase I detoxification (transformation)
Inhibitors of Phase I Transformation
It is known that certain drugs, toxins, foods and herbs can inhibit CYP450 enzymes. Science is in the early stages of figuring out how and which constituents are inhibitory. Please remember that it is usually constituents and not whole herbs or whole foods that are examined.
Many flavonoids have been studied in vitro and in animals showing their ability to lower CYP450 and to increase Phase II enzymes except for SULTs which they inhibit. Naringin has been extensively studied and its reactions well documented in humans.
Naringenin is high in grapefruit juice and less so in oranges and tomato skin.
Other flavonoids that have demonstrated mild inhibition of multiple CYPs in animal models include genistein, diadzein, and equol from soy, and theaflavins from black tea.
Additonal inhibitors are curcumin from turmeric; capsaicin form chili pepper; eugenol from clove oil; quercetin from onions, and goldenseal.Quercetin derivaties isoquercetin and rutin are shown to increase CYP450 and phase II GST and UGT. Some discrepancy on quercetin.
Research on Herbal Constituents
You can not assume a constituent and a whole food/herb will have the same actions. It can make you be cautious and more aware though. For example, Milk thistle has been studied in relation to its effect on CYP450 system. If you look through the research you will find conflicting studies. In vitro studies would make it appear that constituents in milk thistle would inhibit CYP3A, while clinical studies are conflicted and look more like the ingested amount is not large enough to effect the person in a real life situation.
If you look at most of the data (largely in vitro) available it would look at first glance that Milk thistle inhibits a variety of CYP450 enzymes. Here is an overview: Milk thistle components have been shown to inhibit a variety of cytochrome P450 (CYP) isozymes, including CYP3A4, CYP2D6 and CYP2C9; UDP glucuronosyltransferases (UGT), including UGT isoform 1A1 (UGT1A1); and several efflux transporters, including ATP-binding cassette (ABC) transporter B1 (ABCB1, P-glycoprotein.
However, clinical studies look like not enough of the key constituents are ingested to do this. The fact is that they are just looking at constituents also. Not the whole plant. Here are a few synopsis of these studies.
1. Collectively, this study indicates that milk thistle poses little risk of interfering with the pharmacokinetic profile of chemotherapeutic agents that are substrates for CYP3A4 and UGT1A1. (Nielka P.H. van Erp, 2016)
2. So far, the clinical research that I have read which uses standard doses of various Silybum products on the market shows that the products do not reach a threshold strength that would end up causing an inhibitor action in relation to the drugs tested. (Petra Jan?ová,, 2007), (Zuber R, 2002), (van Erp NP, 2005) One clinical study I find did find an issue with it. I include some specifics as follows: There is significant difference in absorption of domperidone on pretreatment with silymarin is due to the inhibition of P-glycoprotein and CYP3A. Silymarin, which inhibits CYP3A4, should be contraindicated for domperidone.(Yamsani sk, 2014)
Currently, the research is inconclusive about if and how various constituents from milk thistle are acting in my humble opinion. The moral of this study is to be very cautious about all research until the results show extremely obvious cause and effects.
Back to talking about inhibition in general:
When there is more than one compound that is transformed by a single 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 due to increased toxic load. I would not really call this inhibition but it does decrease enzymes available. It is simply a case of using up available enzymes faster than they can be made.
Another mechanism of inhibition is depletion of necessary cofactors.
Specific Information on Inhibitors of phase I transformation
Grapefruit juice contains furanocoumarins with the ability to inhibit the metabolism of a variety of drugs. It inhibits CYP3A4. It is thought that CYP3A4 metabolizes about 50% of all drugs. CYP3A4 is located in the epithelial cells of the small intestines and colon and in the parenchymal cells of the liver. So, many drugs are metabolized twice by CYP3A4 before reaching the circulation. In a 2013 research article it was found that more than 85 medications are effected by grapefruit juice. They mentioned that the interaction with 43 of them can cause serious effects such as nephrotoxicity, GI bleeding, depression and other less than fun conditions.
200–250 mL juice or a whole grapefruit has sufficient potency to cause a significant pharmacokinetic interaction. Frequent use over a period of time will increase the action even more. Additionally, there is a greater reaction seen in those older than 70 years.(David G. Bailey, 2013)
I have worked with people who have actually used grapefruit juice to inhibit CYP3A4 on a daily basis so that they could take less of an expensive drug and still get the desired results.
More on Inducers and inhibitors in relation to drugs.
If a medication is taken with an agent that inhibits its metabolism, then the drug level can rise and possibly result in a harmful or adverse effect. If the medication is taken with an agent that increases its metabolism the drug level can fall and also possibly result in a harmful or adverse effect.
Information regarding a drug's CYP450 metabolism and its potential for inhibition or induction can be found on the drug label and accessed through the U.S. Food and Drug Administration (FDA) or manufacturer's websites. There are universities with websites that have lists of inducers and inhibitors of CYP450 in relation to drugs, food and herbs also.
Gender and Age Relationship
The CYP3A family of enzymes is sensitive to hormones. It appears to be regulated partly by progesterone. or the ratio of progesterone to estrogen. In premenopausal women the have 30%-40% more CYP3A4 than men or postmenopausal women. Pregnant women also appear to have higher levels.
Relationship of CYP enzymes to Disease and Health
It is known that impaired liver function can cause lower transformation/detoxification in general. Since the enzymes of Phase I and Phase II are located in different parts of the cell, different liver diseases may effect the activities of enzymes more or less than others. Additionally, the liver makes bile that is used to remove toxins out of the body via the GI tract. The Gall bladder is also important in that it stores and concentrates bile.
I would also think that any disease in the intestines, especially the small intestines which causes inflammation or damage to the epithelial cells will also effect the intestinal transformation/detoxification enzymes. The microbiome can effect both the health of the gut wall as well as nutrients available, so I would think the gut microbiome can easily effect the bodies transformation/detoxification enzymes.
Lack of cell membrane integrity from poor quality of dietary lipids or lack of dietary lipids or oxidation of membrane lipids may also be a factor.
People with a high inducibility phenotype for CYP1A1 have a higher risk for cancer, no matter what their exposure to known carcinogens.
Idiopathic pancreatitis has been associated with upregulation of CYP450 enzymes in many patients. Xenobiotic exposure seems to also increase the susceptibility to alcohol-related pancreatitis.
Studies have identified the occurrence of defective alleles as having four key phenotypes (poor, intermediate, extensive and ultrarapid metabolizers) that could be used to distinguish the diversity in the human CYP450 system.
5-10% of the caucasian population have a CYP2D6 polymorphism. (important for metabolizing a lot of drugs) These people are known as poor metabolizers of many drugs and xenobiotics to different degrees.The people with the worse metabolization have two genes encoding for the lower CYP2D6 activity.
There are also people who are called extensive metabolizers who have greater CYP2D6 activity. These folks are really good at metaoblizing the drugs and xenobiotics that the CYP2D6 enzymes work on. This is no where near as common as the poor metabolizers. CYP2D6 is primarily influenced by genetics so determination of slow or fast metabolism of CYP2D6 is possible with gene analysis.
For the chemistry geeks I have included this:
CYP450 enzymes are so named because they are bound to membranes within a cell (cyto) and contain a heme pigment (chrome and P) that absorbs light at a wavelength of 450 nm when exposed to carbon monoxide.
CYP enzymes have a unique ability to activate molecular oxygen to suandbsequently insert a single oxygen atom stereo-specifically into inert chemical bonds. CYP enzymes catalyses the insertion of oxygen into activated carbon - hydrogen bonds to yield alcohol (e.g. RH + O2 + 2H+ + 2e– → ROH + H2O). However, they can also carry out plethora of other reactions including epoxidation, dealkylation and heteroatom oxidation (Saxena, Parijat, Sudeep, Feroz, 2008)
A specific gene encodes each CYP450 enzyme. Every person inherits one genetic allele from each parent. Alleles are referred to as “wild type” or “variant,” with wild type occurring most commonly in the general population. An “extensive” (i.e., normal) metabolizer has received two copies of wild-type alleles. Polymorphism occurs when a variant allele replaces one or both wild-type alleles. Variant alleles usually encode a CYP450 enzyme that has reduced or no activity. Persons with two copies of variant alleles are “poor” metabolizers, whereas those with one wild-type and one variant allele have reduced enzyme activity. Finally, some persons inherit multiple copies of wild-type alleles, which results in excess enzyme activity. This phenotype is termed an “ultrarapid” metabolizer.
CYP450 enzyme polymorphism is responsible for observed variations in drug response among patients of differing ethnic origins. It is known that 7 percent of white persons and 2 to 7 percent of black persons are poor metabolizers of drugs dependent on CYP2D6, which metabolizes many beta blockers, antidepressants, and opioids. 20% of Asian persons is a poor metabolizer of drugs dependent on CYP2C19, which metabolizes phenytoin (Dilantin), phenobarbital, omeprazole (Prilosec), and other drugs. Variables in drug response among persons of different ethnic origins also can be caused by genetic variations in other drug-metabolizing enzymes, drug transporters, and drug receptors.
Other Phase I Enzymatic Systems
Flavin-containing monooxygenases Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a “soft-nucleophile”, usually nitrogen or sulfur. FMO is a flavoprotein containing a single FAD. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics.
As with the CYP monooxygenase system, over the course of evolutionary plant–animal warfare, FMO developed broad substrate specificity at the expense of turnover. Both monooxygenases are capable of oxidizing thousands of plant alkaloids and other natural products as well as the thousands of synthetic therapeutic drugs. This is an excerpt from this research review which can be found here.
Other enzyme systems are the alcohol and aldehyde dehydrogenases (metabolize alcohol), and monoamine oxidases (metabolize serotonin, dopamine and epinephrine).
Mycotoxins and the CYP450 enzymes that are induced by them
Aflatoxin B1 - CYP3A4, CYP1A2
Expression of this gene is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. The enzyme's endogenous substrate is unknown; however, it is able to metabolize some PAHs to carcinogenic intermediates. Other xenobiotic substrates for this enzyme include caffeine, aflatoxin B1, and acetaminophen.
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. 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 system as well as variations in the cytochrome P450 system are thought to contribute to the differences observed in interspecies aflatoxin susceptibility.
Tidbits of Fun Information
CYP2E1 catalyzes oxidation of ethyl alcohol to acetaldehyde, detoxifies many small carbon-chain molecules such as ketone bodies from gluconeogenesis and the break down of fatty acids. CYP2E1 is increased in insulin dependent diabetes, in morbidly obese people and in starvation.
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