Introduction

Human cytochrome P450 (P450, CYP) enzymes catalyze a great number of metabolic reactions that have important effects on the biological activities (physiologic, therapeutic, and/or toxic) of xenobiotics such as drugs, natural products, general chemicals (e.g., environmental chemicals such as pesticides, pro-carcinogens), and physiological compounds. Their general role and significance for metabolism in humans has been discussed and reviewed previously. In addition, in previous publications efforts were made to estimate the participation of the activity of different groups of enzymes, e.g. oxidoreductase enzymes (FMO (microsomal flavin-containing monooxygenase), AKR (aldo–keto reductase), MAO (monoamine oxidase), and P450 enzymes), in the metabolism of natural products and physiological chemicals and general chemicals in humans. When the groups of chemicals were analyzed, the results showed the highest values for participation of P450 enzymes in the metabolism of drugs and general chemicals as substrates. For P450 enzymes the calculations also showed that, regarding drug metabolism, more than three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, with the largest fraction of the P450 reactions being catalyzed by P450 3A enzymes. Compared to other oxidoreductase enzymes and taking into consideration chemicals that are classified as carcinogens, our calculations showed that metabolic activations of the compounds to toxic metabolites are dominantly catalyzed by P450 enzymes (66% of bioactivations) and that, within this group, six P450s (1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) accounted for 77% of the P450 activation reactions. In the present review we have updated and extended our calculations to general activation reactions forming potentially toxic metabolites as a consequence of metabolic activation of drugs, natural products, physiological compounds, and general chemicals (Rendic 2002; Rendic and Di Carlo 1997; Rendic and Guengerich 2012,2015). We recently reviewed the properties (mechanisms, induction, inhibition, toxic effects, and benefits) of human P450s belonging to the P450 Families 5–51 (i.e., 22 of the total 57 P450s) that are responsible for metabolism and biosynthesis of physiological compounds, including their substrate selectivity, information, and references (Rendic and Guengerich 2018). In the present paper, we update and discuss important aspects of many of the P450s belonging to Families 1–4, including the reactions and the roles in metabolic activation of xenobiotics (drugs, natural products, general chemicals) and physiological compounds.

Results and discussion

A synopsis of the data used for the analysis of the catalytic activity of P450 Families 1–4 is presented in Table 1. Data were collected from more than 5152 references. The total number of data entries for enzymatic reactions catalyzed by P450s belonging to 1–4 Families was 7686 of which 1114 (~ 15%) were defined as bioactivation reactions of different degrees. When considering the activation of all compounds the results show predominant participation of P450s 3A4, 1A2, and 1A1, followed by P450s 2E1 and 1B1. P450s 2C9, 2D6, 2A6, 2C19, and 2B6 also have significant participation in bioactivation reactions (Fig. 1).

Table 1 Number of data entries related to metabolic activation of drugs, general chemicals, natural products, and physiological compounds catalyzed by cytochrome P450 Families 1–4
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Fig. 1
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Participation of human P450 Families 1-4 in activation of all compounds to potentially toxic metabolites (7686 reactions, 1114 activation reactions)

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Data analyzed were divided into four groups of compounds: General Chemicals, Drugs, Natural Products, and Physiological Compounds. Of the 2165 reactions for General Chemicals, 618 (29%) were classified as activations; for 4032 Drugs entries, 237 (6%) were classified as activations; for the 952 reactions under Natural Products, 186 (20%) were classified as activations; for the 530 Physiological Compounds, 75 reactions (14%) were classified as activations (Table 1).

General chemicals

We reported previously that the metabolism of General Chemicals catalyzed by human enzymes is predominately catalyzed by P450 enzymes in humans (~ 92%) (Rendic and Guengerich 2015). Other enzymes, besides P450s, that participate in a greater extent include those in the AKR, FMO, and MAO families (Rendic and Guengerich 2015). P450 enzymes dominate in bioactivation of carcinogens (66%) over other xenobiotic-metabolizing enzymes (Rendic and Guengerich 2012). The present data show that among P450 enzymes, Family 1 enzymes (P450s 1A1, 1A2, B1) dominate in activations of General Chemicals, followed by P450s 2E1, 3A4, and 2A6 (Fig. 2).

Fig. 2
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Participation of human Families 1-4 in activation of general chemicals to potentially toxic metabolites (2165 reactions, 618 activation reactions)

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The following examples illustrate the participation of P450 enzymes in the bioactivation of selected General Chemicals substrates.

Polycyclic aromatic hydrocarbons (PAHs)

Examples (213 data entries) of the metabolic activation of a group of general chemicals (e.g., polycyclic aromatic hydrocarbons (PAHs), heterocyclic and aromatic amines, insecticides, organic solvents) are presented in Table 2. The majority of the data presented (75 data entries) involve PAHs and their metabolites. Of the 76 entries presented in Table 2, 24 are attributed as “high activity” or “high activation” and are catalyzed by P450 1A1, 1A2, 1B1, 2A13, and 2A6 enzymes. These data correlate well with experimental findings on the activation of PAHs by P450 enzymes (Shimada et al. 2013). The parent PAH compounds are not toxic per se but their products formed by hydroxylation and epoxidation reactions, catalyzed by P450 enzymes, are reactive and interact with cellular macromolecules. Consequently, the literature data on activation of PAHs are predominately focused on activation of the PAH metabolites (e.g., dihydrodiols possessing different stereochemical structures) to ultimate toxic dihydrodiol epoxides, as exemplified by the classic activation of benzo[a]pyrene (B[a]P) (Fig. 3).

Table 2 Examples of the metabolic activation of groups of general chemicals (e.g. polycyclic aromatic hydrocarbons, heterocyclic and aromatic amines, insecticides, organic solvents) by human cytochrome P450 enzymes
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Fig. 3
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Activation of benzo[a]pyrene to toxic trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro product (dihydrodiol epoxide) by P450 enzymes

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The following examples are taken from Table 2 illustrate PAH compounds for which metabolic activation is attributed as “high activity reaction” and/or “high activation” (for references see Table 2):

P450 1A1: 5-Methylchrysene, trans-5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene (7,12-DMBA), trans-7,12-DMBA-3,4-diol, (±)-benzo[a]pyrene (B[a]P) -7,8-dihydrodiol, cis-(−)-B[a]P-7,8-dihydrodiol, trans-(+)-B[a]P-7,8-dihydrodiol, trans-(−)-B[a]P-7,8-dihydrodiol, dibenzo[a,l]pyrene (DB[a,l]P), trans-(−)-DB[a,l]P-(11R,12R)-diol

P4501A2: Dibenzo[a,l]pyrene (DB[a,l]P)

P4501B1: B[a]P, (+, −)-B[a]P-7,8-dihydrodiol, cis-(−)-B[a]P-7,8-dihydrodiol, trans-(+)-B[a]P-7,8-dihydrodiol, trans-5-Methylchrysene-1,2-diol, trans-7,12-DMBA-3,4-diol), dibenzo[a,l]pyrene (DB[a,l]P), trans-(−)-DB[a,l]P-(11R,12R)-diol

P450 2A13: trans-5-Methylchrysene-1,2-diol

P450 2A6: trans-5-Methylchrysene-1,2-diol

Heterocyclic and aromatic amines

Activation of heterocyclic, aromatic, and azoaromatic amines is represented by 58 cadsentries (Table 2) of which 15 are attributed as “high activity” and/or “high activation” catalyzed by P450 1A1, 1A2, 1B1, 2A13, 2A6, and 3A4. The reactions of activation or aromatic and heterocyclic amines are presented in Figs. 4 and 5 as illustrated by activation of 2-aminofluorene and MeIQx, respectively.

Fig. 4
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Activation of 2-aminofluorene to toxic N-hydroxy product by P450 enzymes

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Fig. 5
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Activation of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQx) to toxic N-hydroxy product by P450 enzymes

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The following examples illustrate the metabolic activation of heterocyclic compounds by specific P450s (Table 2 and references therein):

P450 1A2: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a,3,2′-d]-imidazole (Glu-P-1)

P450 1A1: 2-Aminoanthracene (2-AA), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 6-aminochrysene

P450 1A2: 2-Aminofluorene (2-AF)

P450 1B1: 2-AA

P450 2A6: 2-AA

P450 2A13: 2-AF

P450 3A4: 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 6-aminochrysene

Insecticides

Activation of organophosphate insecticides is represented by chlorpyrifos, diazinon, parathion, and azinphos-methyl (Table 2 and references therein). The compounds are metabolically activated to neurotoxic metabolites (i.e. oxon derivatives) by desulfuration reactions catalyzed by P450 enzymes . Chlorpyrifos (Fig. 6) and parathion (Fig. 7) are activated by P450 1A2, 2B6, 2D6, 2C8, 2C19, 3A4, and 3A5 enzymes, of which P450 2B6 is the most prominent at lower concentrations (20 µM) and having the highest kcat/Km value. In addition to the oxon derivative, chloropyrifos is also metabolized to the less toxic 3,5,6-trichloro-2-pyridinol by P450 3A4 (Jan et al. 2016; Crane et al. 2012a, 2012b; Croom et al. 2010; Mutch and Williams 2006).

Fig. 6
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Activation of chlorpyrifos to a toxic oxon product by cytochrome P450 enzymes

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Fig. 7
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Activation of parathion to a toxic oxon product (paraoxon) by P450 enzymes

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Fig. 8
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Participation of human P450 Families 1-4 in activation of drugs to potentially toxic products (4039 reactions, 235 activation reactions)

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Azinphos-methyl is activated primarily by P450 1A2 (at low concentrations), and 2B6 and 3A4 (at higher concentrations) (Table 2) (Buratti et al. 2002, 2003). The entries in Table 2 show that at lower concentrations organophosphates are activated predominately by P450 1A1, 1A2, 2B6, and 2C19 and at high concentrations by P450 3A4.

Drugs

A present and historical view of the activation of drugs and their conversion to reactive metabolites as substrates of P450 enzymes has been reviewed recently by one of the authors of the present paper. It has been pointed out that P450 metabolic activity often prevents drug toxicity (for instance making drug elimination faster), but on the opposite side it can, in some cases, result in the conversion of drugs to reactive metabolites that cause toxicity (Guengerich 2020). The final properties of the products of drug-P450 enzyme reactions can also be significantly affected by factors such as (a) variations in the activity caused by genetic polymorphism and thus primarily on the level of single nucleotide variations (SNVs), or (b) by enzyme induction and/or inhibition of activity by environmental chemicals or by co-administered drug(s) (Guengerich and Rendic 2010; Guengerich 2020). Examples of drugs that are converted to toxic metabolites, due to the activity of P450 enzymes, are listed in Table 3. It must be emphasized that most drugs, used in recommended doses, are not or are only slightly toxic per se due to extensive testing in preclinical and clinical testing of drugs. However, as mentioned before, toxic metabolites might be formed under circumstances of enhanced dose, when applied with other drugs/chemicals that might redirect metabolism pathway to the formation of toxic metabolites, or when genetic polymorphism of the particular enzyme was not tested or observed in early drug testing. It is prudent to remember the words of Paracelsus, paraphrased, “the dose makes the poison (only the dose distinguishes a medicine from a poison)” (Borzelleca 2000). Selected examples of drugs taken from Table 3 are discussed, for which toxicity is related to metabolic conversion to toxic products and is known to occur during clinical use. In addition, therapeutic compounds are presented that are used as pro-drugs. Such pro-drugs are therapeutically inactive until activated by P450 enzymes but can became also cytotoxic in healthy cells/tissues when used in therapy (e.g., the anticancer drugs cyclophosphamide, ifosfamide, and AQ4N (banoxantrone; 1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-hydroxy-anthracene-9,10-dione bis-N-oxide)). Also included is the natural product drug ellipticine, which is used in cancer therapy and activated to a cytotoxic metabolite (Table 3). However, while the inherent toxicity of a drug might be lowered, the metabolites formed might be also less toxic and less therapeutically active. An example of such a drug is trabectedin (ecteinascidin 743), an anti-cancer drug of marine origin for which the side effects include myelosuppression, hepatotoxicity, and nausea and vomiting (Held-Warmkessel 2003). Trabectedin is metabolized by P450 3A4 (major enzyme) and in addition by P450s 2C9, 2C19, 2D6, and 2E1. Metabolic and inhibition studies revealed that the metabolites formed are less cytotoxic and less therapeutically active than the parent drug. Inhibitors of P450 enzymes significantly increased cytotoxicity of the drug in a human cell line model system (Reid et al. 2002; Brandon et al. 2005, 2006).

Table 3 Examples of the metabolic activation of drugs by human cytochrone P450 enzymes
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The numbers of activation reactions of drugs as substrates of human P450 enzymes are presented in Fig. 8, calculated from our records. Of the total of 4039 reactions, 235 (~ 6%) involve activation and formation of potentially toxic intermediates or metabolites. P450 3A4 clearly dominated in the formation of toxic metabolites compared with other P450s, catalyzing ~ 25% of the bioactivation reactions.

The following examples illustrate the participation of P450 enzymes in the bioactivation of selected drugs (Table 3).

AQ4N

AQ4N, an aliphatic amine di-N-oxide, is a potent topoisomerase II inhibitor and in clinical trials as a potential anticancer drug. It is inactive until enzymatically bioactivated to an active amine under the reductive conditions present in hypoxic tumor cells (Fig. 9) (Patterson 1993; Lalani et al. 2007; O'Rourke et al. 2008). Because the amine AQ4 is very toxic to normal cells, it is not inherently suitable for delivery as an anticancer drug. AQ4N is reported to be a substrate of several P450s (i.e. 1A1, 1A2, 1B1, 2B6, 2W1, 2S1, and 3A4) (Table 3 and references therein), but most efficiently by P450s 1A1 and 2B6 (Yakkundi et al. 2006)). Under reducing oxygen conditions (hypoxia) AQ4N is reduced to the cytotoxic AQ4-mono-N-oxide (AQ4M) and amine (AQ4) (Patterson et al. 1999). Of the enzymes involved in the metabolism of AQN4, P450 2W1 is highly expressed in some human colon and adrenal tumors and was suggested as tumor-specific enzyme. In addition, strong expression of P450 2S1 has been reported in tumors of epithelial origin and hypoxic tumors and the gene was found to be overexpressed under hypoxic conditions (Karlgren et al. 2005; Saarikoski et al. 2005; Rivera et al. 2007; Nishida et al. 2010; Xiao et al. 2011).

Fig. 9
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Activation of AQ4N to AQ4M and AQ4 by P450 enzymes

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Cyclophosphamide and ifosfamide

Cyclophosphamide and ifosfamide are widely used anticancer agents that require metabolic activation by P450 enzymes (Figs. 10 and 11, respectively). While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic products. Cyclophosphamide and ifosfamide undergo extensive P450-catalyzed metabolism to yield both active (4-hydroxylated) and therapeutically inactive but neurotoxic N-dechloroethyl metabolites, and ovarian toxicity is a major concern with cyclophosphamide therapy. The human liver microsomal P450 metabolism of cyclophosphamide and ifosfamide 4-hydroxylation is well characterized (Table 3 and references therein). Of all P450 enzymes, P450 3A4 exhibited the highest N-dechloroethylation activity (bioactivation) toward both cyclophosphamide and ifosfamide, whereas P450 2B6 and P450 3A4 displayed high N-dechloroethylation activity toward ifosfamide but not cyclophosphamide. With cyclophosphamide as substrate, P450 3A4 was shown to catalyze ≥ 95% of liver microsomal N-dechloroethylation, whereas with ifosfamide as substrate, P450 3A4 catalyzed an average of ~ 70% of liver microsomal N-dechloroethylation (range 40–90%), with the balance of this activity catalyzed by P450 2B6 (range 10–70%, depending on the P450 2B6 content of the liver) (Huang and Waxman 2000). In the case of cyclophosphamide treatment, determination of selected P450 enzyme genotypes (such as frequencies of the variant alleles CYP2B6*5, CYP2C19*2, CYP2C9*2, and CYP3A5*3) has been proposed for predicting the risk of premature ovarian failure in lupus nephritis patients (Takada et al. 2004).

Fig. 10
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Activation of CPA to acrolein and phosphoramide mustard by P450 enzymes

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Fig. 11
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Activation of IFA to N-dechloroethyl metabolites by P450 enzymes

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Acetaminophen

Acetaminophen (paracetamol, Tylenol®) is one of the most widely used drugs in the world. It is very safe when used at therapeutic doses. However, it is also involved in ~ ½ of the cases of drug-induced liver failure and well exemplifies the axiom of Paracelsus about the “the dose makes the poison” (Larson et al. 2005). When overdosed, the drug causes mitochondrial dysfunction and centrilobular necrosis in the liver in humans and experimental animals (Yoon et al. 2016). Normally most of the ingested drug is eliminated by glucuronidation and sulfation, catalyzed by UDP-glucuronosyltransferases (UGT1A1 and 1A6) and sulfotransferases (SULT1A1, 1A3/4, and 1E1), respectively, producing nontoxic conjugates (McGill and Jaeschke 2013) (Fig. 12).

Fig. 12
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Activation of acetaminophen (paracetamol) to N-acetyl-p-benzoquinone imine by P450 enzymes

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The key mechanism in the hepatotoxicity is P450-catalyzed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which depletes hepatic glutathione and accumulates to cause hepatocellular liver damage, including oxidative stress (Fig. 13). Approximately 5–9% of orally administered acetaminophen is metabolized by P450 2E1, 1A2, and 3A4 catalyzed oxidation reactions (Table 3 and references therein). At a toxic concentration the formation of the NAPQI-glutathione conjugate was highest with P450 3A4, having the lowest Km value of 130 μM (for comparison therapeutic concentrations of paracetamol are ~ 50 μM and toxic concentrations are ~ 1 mM) followed by P450 2E1 and P450 1A2 (Patten et al. 1993)). It has been proposed that human P450 3A4 is the major enzyme catalyzing acetaminophen oxidation to NAPQI (Laine et al. 2009). Other studies using inhibition studies with human recombinant enzymes indicated that P450 1A2 is the enzyme responsible for acetaminophen metabolic activation (Tan et al. 2008). In mice, the deletion of P450 2e1 or 1a2 blocks acetaminophen toxicity (Lee et al. 1996; Zaher et al. 1998).

Fig. 13
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Activation of halothane by reductive and oxidative dehalogenation by P450 enzymes

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Halothane

Halothane was previously the most widely used anesthetic agent and in 1963 was reported to cause liver necrosis in humans. It was shown that halothane liver necrosis was induced following pretreatment of rats with polychlorinated biphenyls, known inducers of P450 enzymes. The liver necrosis caused by halothane anesthesia could be prevented by administration of metyrapone, a rather non-selective inhibitor of P450 enzymes, prior to anesthesia. These findings indicated that halothane toxicity resulted from metabolic activation of halothane by P450 enzymes (Nastainczyk et al. 1982). In addition, halothane hepatotoxicity could be potentiated in rats by chronic administration of ethanol, an inducer of P450 2E1 (Takagi et al. 1983). Human P450s activate halothane by both reductive and oxidative metabolism (Table 3; Fig. 13), and oxidative dehalogenation by P450 2E1 is a major in vivo reaction with low Km values (and also results in lipid peroxidation). Limited participation of P450 2A6 activation has been indicated in vivo. Reductive activation of halothane is catalyzed by P450 2A6 and 3A4 enzymes (Table 3 and references therein). Halothane oxidation, the major metabolic pathway, leads to the production of the reactive electrophile trifluoroacetyl chloride, and subsequent acylation of liver proteins results in the formation of trifluoroacetylated protein neoantigens. Metabolic halothane reduction leads to the formation of the 2-chloro-1,1,1-trifluoroethyl radical by hemolytic cleavage of the C–Br bond (Fig. 13) (Kurth et al. 2014). Halothane has been replaced in most countries by other, less toxic, inhalation anesthetics due to its induced hepatitis, but there is still the possibility that it is in use in some developing countries.

Fig. 14
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Activation of 17α-ethynylestradiol by aromatic hydroxylation to catechol metabolites by P450 enzymes

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17α-Ethynylestradiol

17α-Ethynylestradiol is in use as the estrogenic component of oral contraceptives (Bolt 1979). Similar to natural estrogens (see the Physiological Compounds section, vide infra) (Lacassagne 1932), 17α-ethynylestradiol is a weak carcinogen in rats, and carcinogenic activity has been associated with the formation of catechol metabolites (Fig. 14) (Zhu et al. 1993). C2-hydroxylation catalyzed by P450 enzymes (mainly P450 3A4) was found to be the main metabolic pathway of 17α-ethynylestradiol in individuals using oral contraceptives (Guengerich 1988)). This metabolite is excreted as 2-methoxyethynylestradiol after O-methylation (Back et al. 1984). Induction of P450 3A4 by rifampicin, barbiturates, or herbal remedies such as St. John’s wort can lead to increased clearance and unplanned pregnancy (Bolt et al. 1977; Guengerich 1988).

Natural products

Natural products, including herbal supplements, can have multiple effects on the activity of P450 enzymes, for instance inhibition or induction of activity and/or their expression (St. John’s wort, vide supra). By changing activity and/or expression of the enzymes and applied concomitantly with drugs, natural chemicals can provoke drug-phytochemical interactions. Such activity might result in altered therapeutic and or toxic properties of drugs (Guengerich and Rendic 2010; Rendic and Guengerich 2010). Examples of different classes of natural compounds that can be activated to toxic metabolites by cytochrome P450s (e.g., alkaloids, monoterpenes, mycotoxins, N-nitrosamines) are presented in Table 4. Natural products, as substrates of P450 enzymes, can be both activated to toxic and detoxicated to nontoxic products by P450 enzymes in different ways. For instance, aflatoxin B1 (AFB1) is activated to toxic and detoxicated to nontoxic metabolites by oxidative reactions, while aristolochic acid is activated by nitro reduction under (partially anaerobic conditions), and oxidative metabolism results in the formation of a nontoxic O-demethylated product. Estragole and safrole are examples in which metabolism by P450 enzymes to nontoxic metabolites can be followed by activation to toxic metabolites by conjugation to form a sulfate ester (Table 4 and references therein).

Table 4 Examples of metabolic activation of natural products by human cytochrome P450 enzymes
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3-Methylindole (skatole) is formed in nature by microbial degradation of tryptophan and tyrosine (Carlson and Breeze 1984), but is also present in humans where it is formed by the decarboxylation of tryptophan in the large intestine. 3-Methylindole is a selective pulmonary toxicant and, in addition to intestinal formation and absorption, cigarette smoke is an additional source of 3-methylindole in smokers. 3-Methylindole may provoke pneumotoxicity and lung cancer by the activity of P450 1A1 and P450 2F1 (Weems et al. 2010). Toxicity of 3-methylindole depends on bioactivation by several reactions: epoxidation (3-methyloxindole formation, P450 1A1, 1A2, 1B1, 2E1, 2A6), C-hydroxylation (indole-3-carbinol formation, P450 1A1, 1A2, 1B1), and dehydrogenation (3-methyleneindolenine formation, P450 1A1, 1A2, 2A13, 2F1 (Table 4 and references therein).

The numbers of activation reactions catalyzed by human P450 enzymes reacting with natural products as substrates are presented in Fig. 15. Of the total of 952 reactions identified in our records, 152 (~ 16%) involve bioactivation and the formation of potentially toxic products. The major P450s involved in the activations are P450s 1A2 (~ 12%), P450s 2E1 and P450 3A4 (~ 11% each), followed by P450 1A1 and 2A6 (~ 10%).

Fig. 15
figure 15

Participation of human P450 Families 1-4 in activation of natural products to potentially toxic metabolites (952 reactions, 186 activation reactions)

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The following examples illustrate the participation of P450 enzymes in the bioactivation of selected natural compounds (Table 4).

Aflatoxins

AFB1 is a potent hepatocarcinogen in animal models and also classified as a hepatocarcinogen in humans. AFB1 is metabolically activated by P450 enzymes to form cytotoxic and DNA-reactive intermediates (Fig. 16). AFB1 is activated to the toxic exo-8,9 epoxide most prominently by P450 Subfamily 3A enzymes in liver and P450 2A13 in the lung (Shimada and Guengerich 1989; Deng et al. 2018). In addition to its hepatotoxicity, AFB1 can be toxic in lungs (at least in animal models) due to the activity of P450 2A enzymes. P450 3A enzymes (3A4 and 3A5) oxidize AFB1 to the highly mutagenic exo-8,9-epoxide (Fig. 16), while P450 1A2 oxidizes it to a roughly equimolar mixture of toxic exo- plus the endo-epoxide, the latter of which is essentially non-mutagenic (Iyer et al. 1994). Both P450 3A4 and 1A2 enzymes also catalyze AFB1 detoxication reactions, i.e. 3α-hydroxylation in the case of P450 3A4 (aflatoxin Q1 formation) and 9a-hydroxylation in the case of P450 1A2 (aflatoxin M1 formation) (Rendic and Guengerich 2012)). This example illustrates that P450 enzymes can catalyze both activation and detoxication reactions acting on the same substrate. The toxic AFB1-exo-8,9-epoxide is detoxicated by glutathione (GSH) transferases by conjugation of GSH to the epoxide (Johnson et al. 1997; Deng et al. 2018; Yang et al. 2012). In addition to a being substrate of P450 enzymes, AFB1 is an inducer of P450 1A1, 1B1, and 3A4 in monocytes (Bahari et al. 2014), and the compound might enhance its own metabolism or metabolism of another substrate of the enzyme.

Fig. 16
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Activation (formation of 8,9-exo-epoxide) and detoxication (formation of AFQ1) of AFB1 by P450 3A4

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Artistocholic acid

Aristolochic acids constitute a group of compounds found naturally in many types of plants known as Aristolochiaceae, including Aristolochia and Asarum (wild ginger) grown worldwide. Aristolochic acid I and II are the predominant chemical toxins in the plants. Aristolochic acid compounds were shown to be the cause of a kidney disease called Chinese herb nephropathy, now renamed aristolochic acid nephropathy (Arlt et al. 2002; Schmeiser et al. 2009; Gökmen et al. 2013). Aristocholic acid is classified by the International Agency for Research on Cancer as a Group I carcinogen. This natural product has also been implicated in the development of another kidney disease, Balkan endemic nephropathy, and its associated urothelial malignancy. The disease is endemic in certain rural areas of Balkan countries located closed to the tributaries of the Danube river basin (Arlt et al. 2007; Grollman et al. 2007; Han et al. 2019). As already mentioned, aristolochic acid I is activated by reduction of the nitro group (under partially anaerobic conditions), and oxidative metabolism results in the formation of nontoxic O-demethylated metabolites. Nitro reduction of aristolochic acid I, considered as the major factor causing its toxicity, is required to exert its carcinogenic properties. The reaction catalyzed by P450s 1A1 and 1A2 results in the generation of N-hydroxyaristolactam I, which leads to the formation of a cyclic acyl nitrenium ion, the intermediate that either forms DNA adducts or rearranges to 7-hydroxyaristolactam I (Fig. 17). Aristolochic acid I oxidation to a nontoxic metabolite by O-demethylation of the methoxy group is catalyzed by the same enzymes, i.e. P450 1A1 and 1A2, with contribution from P450 2C9 and 3A4 (Table 4). The product of the reactions is 8-hydroxyaristolochic acid I, a detoxication product. The O-demethylated metabolite is excreted either in its free or conjugated form (Chan et al. 2006; Shibutani et al. 2010; Arlt et al. 2011; Stiborová et al. 2012a).

Fig. 17
figure 17

Activation of aristolochic acid I and II by P450 enzymes

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Estragole

Estragole is a common component of herbs and spices and is a natural constituent of basil oil. It is also a genotoxic hepatocarcinogen in rodents, and its potential toxic effect in humans is still under consideration. One of the major sources of human exposure to this phytochemical is Foeniculum vulgare Mill. (fennel) (Levorato et al. 2018). Toxicity is ascribed to its hydroxylation in position C1´, catalyzed by P450s 1A2, 2A6, 2C19, 2D6, and 2E1, of which P450s 1A2 and 2A6 are the major enzymes (Table 4 and references therein). Other enzymes can also contribute but at relatively high concentrations of estragole. The metabolite of P450 oxidation is not inherently toxic; however, C1´-hydroxylation of estragole is the first step in activation followed by sulfate conjugation by a sulfotransferase to produce genotoxic 3´-sulfoxyestragole (Fig. 18) (Monien et al. 2019).

Fig. 18
figure 18

Activation of estragole by P450 enzymes and sulfotransferases

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Ethanol

Ethanol is widely consumed and is metabolically activated to toxic acetaldehyde (Fig. 19). The metabolism and activation of ethanol is primarily catalyzed by alcohol dehydrogenase and, to a lesser extent, catalase but under certain circumstances (e.g., high doses) P450 enzymes can also be involved. Many P450 enzymes in Families 1–4 oxidize ethanol to acetaldehyde at high concentrations, namely 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 (Table 3), but P450 2E1 has the highest catalytic activity (i.e., specificity constant, kcat/Km).

Fig. 19
figure 19

Activation of ethanol to toxic acetaldehyde by P450 enzymes

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The role of P450 2E1 in ethanol metabolism has been reviewed recently. P450 2E1, 3A4, and 1A2 were reported as P450s that are significantly involved in the oxidation of ethanol to acetaldehyde under conditions of high concentration (Km ~ 10 mM) and chronic use (Hamitouche et al. 2006; Guengerich and Avadhani 2018; Guengerich 2020).

Safrole

Safrole is a natural compound categorized as an IARC Group 2B carcinogen. It is extracted from sassafras oil or certain other essential oils and also from betel quid. Safrole was reported to be a rodent hepatocarcinogen, and DNA adducts were identified in liver samples of patients having a history of betel quid chewing (Bolton et al. 1994; Chung et al. 2008). In addition, betel quid chewing is associated with oral and hypopharynx cancers (Shield et al. 2017; Chen et al. 2017). The metabolism of safrole was reported to be predominantly catalyzed by P450 1A2, with minor contributions by P450 2E1. It was suggested that the ortho-quinone metabolite may mediate safrole hepatotoxicity (Fig. 20; Table 4 and references therein). Safrole can also, as in the case of estragole, undergo bioactivation by sequential 1´-hydroxylation and sulfation, resulting in reactive intermediates capable of forming DNA adducts (Jeurissen et al. 2004). In addition, it has been reported that safrole is a mechanism-based inhibitor of P450 1A2 (Hu et al. 2019; Yang et al. 2018). It has been also reported that safrole induced P450 2A6 activity and tobacco specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolic activation, resulting in higher NNK-induced genotoxicity (Tsou et al. 2019).

Fig. 20
figure 20

Activation of safrole by P450 enzymes and sulfotransferases

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N´-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The tobacco-specific nitrosamines NNN and NNK are potent carcinogens in animal models and are believed to be causative agents for esophageal cancer in smokers and those using chewing tobacco and snuff. Metabolic activation of NNN is required to exert carcinogenic potential (Fig. 21) and occurs through P450 catalyzed 2´- and 5´-hydroxylation, which generates unstable metabolites that decompose to 4-hydroxy-1-(3-pyridyl)-1-butanone ('keto alcohol') and 4-hydroxy-4-(3-pyridyl)butanal, respectively. The latter cyclizes to 5-(3-pyridyl)-2-hydroxytetrahydrofuran ('lactol'). P450s 2E1, 2A6, and 3A4 were identified as major catalysts for NNN 5´-hydroxylation in human liver microsomes (Yamazaki et al. 1992; Hecht 1998; Wong et al. 2005a; Patten et al. 1996, 1997; Carlson et al. 2016; Fan et al. 2019; Staretz et al. 1997; Fujita and Kamataki 2001a).

Fig. 21
figure 21

Activation of NNN (formation of  DNA adducts) by P450 enzymes

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NNK, a potent tobacco-specific carcinogen, has been demonstrated to induce lung tumors in animals and is suspected to be a human carcinogen. P450s are the major enzymes responsible for the activation of NNK in lung and liver microsomes of rats and mice, as well as in the human liver. Human P450s 2A6 and 3A4 are involved in the activation of NNK (Smith et al. 1995; Staretz et al. 1997). In addition, it was demonstrated that P450s 1A2, 2A6, and 3A4 may be important for the activation of NNK to a DNA-methylating agent (‘keto aldehyde’) via the α-methylene hydroxylation pathway (Fig. 22). P450s 1A2, 2E1, and 2D6 are selective for α-methyl hydroxylation of NNK, leading to keto alcohol and a DNA-pyridyloxobutylating agent. P450 1A2 exhibits at least twice the specificity toward NNK bioactivation compared to P450 2E1 and catalyzed the formation of both, keto alcohol and 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde) with the keto alcohol being the major product (Patten et al. 1996, 1997; Krishnan et al. 2009; Smith et al. 1996) (Table 4 and references therein).

Fig. 22
figure 22

Activation of NNK (formation of DNA adducts) by P450 enzymes

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Physiological compounds

Physiological substrates of P450 Family 1–4 enzymes include eicosanoids, estrogens (e.g., estradiol), fatty acids (e.g., arachidonic acid), cholesterol, fat-soluble vitamins (e.g., vitamins A, D3, E, and K), neurotransmitters (serotonin, tryptamine), leukotrienes, prostaglandins, fatty acids (e.g. arachidonic acid), bile acids (e.g. lithocholic, deoxycholic, cholic acid), corticosteroids, androgens (e.g., androstenedione, testosterone, dihydrotestosterone), and progesterone. In addition to being substrates of P450s Families 1–4, these compounds are predominately substrates of the enzymes belonging to Families 5–51 (with 22 enzymes) (Rendic and Di Carlo 1997; Rendic and Guengerich 2018). The data presented in Fig. 23 show the participation of Family 1–4 P450s in the activation of physiological compounds to some potentially toxic products. Of the total 530 metabolic reactions (data from our records), 75 (14%) involve bioactivation. The highest involvement is with P450s 1A1, 1A2, 1B1, 3A4, and 3A5 (~ 9% each), followed closely by P450 2C9 (8%). Physiological substrates in activation reactions include estrogenic hormones (17β-estradiol and estrone) and fatty acids (Table 5). In addition to being activated to toxic products, fatty acids (exemplified by arachidonic acid) can both down-regulate (Palacharla et al. 2017) or induce P450 activity by changing their expression (Finn et al. 2009). In some cases, the reaction products are not inherently reactive but may have deleterious signaling properties [e.g., 20-HETE, EETs (Sausville et al. 2018)].

Fig. 23
figure 23

Participation of human P450 Families 1-4 in metabolic activation of physiological compounds to potentially toxic metabolites (530 reactions, 75 activation reactions)

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Table 5 Examples of metabolic activation of physiological compounds by human cytochrome P450 enzymes
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Although a relatively low number of activations are ascribed to P450 enzymes interacting with physiological compounds, some of them are important because they can possibly cause either cancer (e.g., estrogenic hormones) or have an important impact on physiological processes related to high blood pressure (arachidonic acid).

17β-Estradiol and estrone

Estrogenic hormones (e.g., 17β-estradiol and estrone) can induce tumors in various organs of experimental animals (Lacassagne 1932). In humans, elevated circulating estrogen levels increase the risk of breast and uterine cancer. Estrogens can act as hormone stimulating cell proliferators and also as procarcinogens, inducing genetic damage (Yager 2000; Liehr 2000). 17β-Estradiol and estrone are eliminated from the body by metabolic conversion to inactive metabolites that are excreted in the urine and/or feces following oxidations and conjugation reactions. The first step in the metabolism of estrogens is hydroxylation catalyzed by P450 enzymes (Fishman et al. 1970; Zhu and Lee 2005). A large number of hydroxylated metabolites are formed and catalyzed by P450 Family 1–4 (Table 5); however, we focus here on reactions leading to the formation of activated and toxic metabolites. Activations of 17β-estradiol and estrone by hydroxylation at positions C2 and C4 have been suggested to be major reactions involved in mammary carcinogenesis and other cancers (Cavalieri and Rogan 2006; Cavalieri et al. 2006). The data (Table 5 and references therein) also show that formation of the major metabolite of 17β-estradiol, 2-hydroxyestradiol, is mainly catalyzed by P450s 1A2 and 3A4, and by P450 1A1 in extrahepatic tissues. P450 1B1, which is highly expressed in estrogen target tissues including mammary, ovary, and uterus, selectively catalyzes the 4-hydroxylation of 17β-estradiol (Guengerich et al. 2003; Chun and Kim 2016; Wen et al. 2007) Formation of catechols of estrone and estradiol is considered as a part of the carcinogenic process, in that these compounds can readily be further oxidized to reactive quinones, semiquinones, and reactive oxygen species are formed (Bolton and Thatcher 2008). 4-Hydroxyestradiol can generate free radicals from redox cycling, with the formation of corresponding semiquinone and quinone forms causing cellular damage. Local formation of 4-hydroxyestradiol in breast and endometrial cancers has been reported (Tsuchiya et al. 2005; Hayes et al. 1996; Spink et al. 1997; Liehr 2000; Shimada et al. 1999; Bolton 2002; Bolton and Thatcher 2008; Fussell et al. 2011). Estradiol-3,4-quinone is more reactive with DNA than estradiol-2,3-quinone, and the relative reactivities of estradiol-3,4-quinone and estradiol-2,3-quinone to form depurinating adducts have been correlated with the carcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens 4-hydroxyestradiol and 2-hydroxyestradiol (Zahid et al. 2006).

Numerous P450s have been detected in breast tumor or adjacent tissue, including P450s 1A1, 1B1, 2A5, 2B6, 2C9, 2D6, 2E1, 2J2, 2S1, 2U1, 3A4, 3A5, 3A43, 4A11, 4V2, 4X1, 4Z1, 26A1, and of course 19A1 (Hellmold et al. 1998; Huang et al. 1996; Iscan et al. 2001; Schmidt et al. 2004). Of these, three enzymes are involved to a major extent in estradiol hydroxylation (i.e. P450s 1A1, 1B1, and 3A4) (Fig. 24). P450 2C9 is also involved in the conversion of both estradiol and estrone, with low activity in forming C4- and C16-hydroxylated products (Table 5). P450 enzymes involved in estrogen metabolism are expressed in both tumor and non-tumor breast tissue; however, higher levels of P450 1B1 and 3A4 were found more often in non-tumor tissue than in tumor tissue. It has been suggested that local activation of estrogen to potentially reactive metabolites by the P450s in breast tissue may play a role in initiating and promoting the carcinogenic process (Modugno et al. 2003).

Fig. 24
figure 24

Activation of 17β-estradiol and estrone (C2- and C4- hydroxylation) by P450 enzymes

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In breast tumor cells, P450 1A1 and 1B1 mRNA levels and rates of both estradiol 2- and 4-hydroxylation were elevated following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Spink et al. 1998). In addition, the inhibitory effects of ketoconazole, cyclosporin A, and cimetidine (inhibitors of P450 enzymes) toward P450 3A4-catalyzed estradiol 2-hydroxylation were reported, and the IC50 values were 7 nM, 64 nM, and 290 µM, respectively (Satoh et al. 2000). It was also reported that non-ortho-substituted polychlorinated biphenyl congeners can, depending on the structure, induce or inhibit P450 1B1 and 1A1 activity and consequently that they might affect the formation of 2- and 4- hydroxylated metabolites of estradiol and the potential for mammary tumorigenesis (Spink et al. 2002a; Pang et al. 1999). Resveratrol was reported to strongly inhibit the TCDD-induced aryl hydrocarbon receptor DNA binding activity, the expression of P450 1A1 and 1B1, and P450 1A1 and 1B1 catalytic activities in MCF-10A breast cancer cells. Resveratrol also reduced the formation of 2- and 4-hydroxyestradiol from 17β-estradiol by recombinant human P450s 1A1 and 1B1, respectively. Furthermore, resveratrol significantly attenuated intracellular reactive oxygen species formation and oxidative DNA damage, and the cytotoxicity induced by the catechol estrogens (Chen et al. 2004). In addition to chemicals that induce or inhibit the activity of P450 enzymes, genetic variation of the enzymes (e.g., P450 1B1) can also affect the metabolic activation and carcinogenesis of 17β-estradiol and estrone, although the effects have not been shown to be large (Shimada et al. 1999; Watanabe et al. 2000). Changes in the expression levels of estrogen-metabolizing P450s not only alter the activity of substrates but may also have physiological effects in liver and target tissues (Chun and Kim 2016).

Arachidonic acid

Arachidonic acid metabolites are key mediators involved in the pathogenesis of numerous cardiovascular, pulmonary, inflammatory, and thromboembolic diseases. Thromboxane A2 is produced by the action of thromboxane synthase (P450 5A1) on the prostaglandin endoperoxide H2 (PGH2), a product of the enzymatic transformation of arachidonic acid by the cyclooxygenases (Rendic and Guengerich 2018). Arachidonic acid is metabolized in a number of tissues (liver, kidney, lung, brain, and the vasculature) by P450 enzymes that form hydroxyeicosatetraenoic acids (HETEs) or epoxides (epoxyeicosatrienoic acids, EETs) (Fig. 25). The reactions occur in different organs (brain, kidney, lung, vasculature, liver). EETs and HETEs have different biological properties, based on sites of production, and can be stored in tissue lipids and released in response to hormonal stimuli.

Fig. 25
figure 25

Activation of arachidonic acid to 20-HETE and EETs by P450 enzymes

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20-HETE has both pro- and anti-hypertensive actions that result from modulation of vascular and kidney function. 20-HETE is a potent vasoconstrictor, and upregulation of the production of this compound can contribute to the elevation of endothelial dysfunction and the increase in peripheral vascular resistance associated with some forms of hypertension. In kidney, 20-HETE exerts anti-hypertensive action by inhibiting sodium reabsorption by the kidney in both the proximal tubule and thick ascending limb of Henle (Williams et al. 2010; Garcia et al. 2017; Zhang et al. 2018; Roman 2002). Formation of 20-HETE is catalyzed by human P450s 4A11, 4F2, and F3B and the epoxygenation of arachidonic acid to EETs is catalyzed by P450s 2C8, 2C9, 2C19, and 2J2 and (to a much lesser extent) by P450 2W1 (Table 5 and references therein). The arachidonic acid products 20-HETE and EETs compose a group of compounds that participate in the regulation of liver metabolic activity and hemodynamics, may be involved in abnormalities related to liver diseases (e.g., cirrhosis), and play a key role in the pathophysiology of portal hypertension and renal failure (Sacerdoti et al. 2003). Arachidonic acid, as a model for metabolic activation of polyunsaturated fatty acids, produced a concentration- and time-dependent toxicity to Hep G2-MV2E1-9 cells, which express P450 2E1, proposed to be related to reactive oxygen intermediates and lipid peroxidation (Chen et al. 1997).

Concluding remarks

The data on activation of xenobiotics and endobiotics catalyzed by P450 enzymes in Families 1–4 are divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds. The metabolites formed are direct toxicants reacting with cell macromolecules in many cases. However, in selected cases the metabolites are not direct toxicants but participate as substrates in additional metabolic reactions (e.g., conjugation reactions) and the resulting products are final toxicants (e.g., estragole). In other cases, the product elicits physiological responses through indirect biological activities (e.g., 20-HETE, EETs). We have emphasized the observed higher number of activations of three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the lower fraction of Physiological Compounds involved as substrates in activation reactions catalyzed by P450 enzymes belonging to Families 1–4, exemplified by estrogen hormones and arachidonic acid. In the group of General Chemicals, P450s 1A1, 1A2, and 1B1 are dominant in the formation of activated metabolites, followed by P450s 3A4 and 2E1 (Fig. 2); in the group Drugs (Fig. 9) P450 3A4 dominates in the formation of activated metabolites. In the group of Natural Products, P450s 1A2, 3A4, and 2E1 dominate in the formation of activated metabolites, followed by P450s 1A1 and 2A6 (Fig. 16); in the group of Physiological Compounds there was no clearly dominant P450 but the highest number of activations is attributed to P450s 1A, 1B1 and 3A (Fig. 23). The results show that Physiological Compounds are substrates infrequently in bioactivation reactions catalyzed by P450 enzymes belonging to Families 1–4, with the exception of estrogens and arachidonic acid.

The results presented to give information on the enzymes that dominate in the bioactivation of specific group of chemicals and might be used as a guide on which enzymes to direct research when testing their bioactivation to toxic metabolites.