*2.1. Small Intestine*

CYP3A4 is the main CYP isoform in the small intestine, accounting for roughly 82% of CYP enzymes expressed in gut tissue [8]. Many drugs, which are 3A4 substrates, have low oral bioavailability due in part to CYP-mediated intestinal first pass metabolism [8]. Due to CYP3A4's broad substrate specificity and high expression in gut tissue, certain foods and dietary supplements can cause significant drug interactions. Grapefruit juice, a potent inhibitor of intestinal 3A4, is known to increase the plasma concentration of common 3A4 substrates, e.g., statins, calcium channel blockers, protease inhibitors, and many others [54]. Inhibition by grapefruit juice does not affect hepatic 3A4 but may decrease intestinal 3A4 function by 62% [32].

#### *2.2. Kidneys*

It has been estimated that the human kidney contains anywhere from 4–20% of hepatic CYP protein content [8]. Only CYPs 2B6 and 3A5 have been confirmed in the human kidney, and evidence for CYPs 3A4, 2C9, and 2C8 is equivocal [6]. Of these, CYP3A5 is the most prevalent isoform [19]. Renal CYP3A5 is highly polymorphic [19]. In fact, genetic variations in renal 3A5 expression may influence nephrotoxicity associated with the immunosuppressive agent, tacrolimus [55], as well as with the anticancer agent, ifosfamide [56]. Further, CYP3A forms are expressed consistently in renal cancer cells and may be involved in renal cancer development and multidrug resistance [57]. Nevertheless, renal CYP3A enzymes may also help suppress cancer via bioactivation of certain agents, forming metabolites that are cytotoxic to tumor cells and benign in noncancer cells [57].

#### *2.3. Lungs*

Lung tissue expresses CYPs 1A1, 1B1, 2A6, 2E1, 3A4, and 3A5 among others [8]. Many respiratory tract CYPs are linked to bioactivation of the constituents of cigarette smoke and enhanced toxicity and carcinogenicity. CYP1A1 is mostly expressed in smokers [8]. Both CYPs 1A1 and 1B1 isoforms are induced by compounds found in tobacco smoke, particularly Benzo(a)pyrene (Bap) [10,58]. Bap is a polycyclic aromatic hydrocarbon (PAH) carcinogen that is converted to DNA-reactive intermediates in a process dependent on CYP1A1 and CYP1B1 metabolic pathways [10]. CYP2A6 is mostly expressed in the trachea and is also thought to be involved in bioactivation of carcinogens from tobacco smoke [8]. CYP2A6 mainly metabolizes nicotine, the primary constituent in cigarette smoke, into cotinine and nicotine-derived nitrosamine ketone (NNK) [59]. Of note, CYP2A6 polymorphisms are involved in the development of lung cancer and nicotine dependence [8,60].

#### *2.4. Heart*

Multiple CYP enzymes relevant to drug metabolism or bioactivation are present in cardiac tissue in low or moderate amounts, including CYPs 1A1, 1B1, 2C8, 2C9, 2D6, 2E1, and 3A4 [31]. Interestingly, higher CYP mRNA expression (e.g., 2D6, 2C) in right ventricular tissue vs. left ventricular tissue, which is indicative of increased drug inactivation at this site, has led some researchers to suggest that differential expression may contribute to therapeutic failure in pharmacological treatment of right ventricular hypertrophy [45]. However, the most prevalent CYP isoform in the heart is CYP2J2, an enzyme involved in metabolizing the anticancer drug, doxorubicin [31]. One common adverse effect of doxorubicin is cardiotoxicity, an effect that may be mitigated in cases of CYP2J2 over-production in cardiomyocytes [61]. Further, cardiac CYPs 1A1 and 2J2 have been shown to be induced in mice following treatment with cocaine and Bap [62].

#### *2.5. Blood*

CYP mRNA and protein, including that of CYPs 3A4, 2A6, 2E1, 1A1, and 1B1, have been observed in human monocytic and lymphocytic cells [13,21,22,50,51], with 2A6 being the most abundantly expressed isoform in monocyte-derived macrophages [21]. Monocytes are part of the mononuclear phagocyte system, a family of myeloid lineage that also includes macrophages and dendritic cells [63]. These blood cells are critical in host defenses against pathogens, as well as in maintaining tissue homeostasis [64]. Blood monocytes may mature into macrophages under inflammatory conditions and migrate to tissues, where they can synthesize and secrete inflammatory mediators [63]. CYP2A6 induction has been observed in monocytes derived from the plasma of smokers [50], and 2A6-mediated metabolism of nicotine is associated with increased oxidative stress and DNA damage in monocytic cells [13,50]. Similarly, cigarette smoke condensate (CSC) induced CYPs 1A1 and 1B1 in in vitro monocyte studies [51]. In addition, alcohol-inducible CYP2E1, which is known to metabolize alcohol in the liver, was also found to be expressed and induced by alcohol in monocytes/macrophages, leading to oxidative stress [21].

#### *2.6. Brain*

Total CYP protein content in the brain is substantially lower compared to the liver and is estimated at 0.5%–2% of hepatic CYP content [65]. Although the contribution to systemic drug metabolism may be minimal, brain CYP activity and variation may have a significant impact on local metabolism and the therapeutic efficacy of centrally acting drugs, including antidepressants, antipsychotics, drugs of abuse, and carcinogens [66]. CYP enzymes are differentially expressed in some regions of the brain and in neurons and glial cells. The highest CYP content is found in the brain stem and cerebellum, and the lowest in the striatum and hippocampus [67]. The proposed functions of cerebral CYPs vary by cell type and location.

#### 2.6.1. Neurons

Drug metabolizing CYPs 1A1, 2B6, 2E1, and 3A4 are found primarily in neurons [65], while CYP2D6 is expressed in pyramidal neurons in addition to glial cells [68]. CYP3A4 is involved in psychotropic drug metabolism, including antiepileptic metabolism [65,68]. Considering that alcohol dehydrogenase (ADH) is not expressed in the brain, while CYP2E1 is constitutively expressed in various brain regions, it is probable that CYP2E1 is the brain's major alcohol metabolizing enzyme [69].

#### 2.6.2. Monocytes and Glial Cells

CYPs are abundant in astrocytes at the blood–brain barrier (BBB), aiding in the regulation of xenobiotic influx into the CNS, blood flow modulation, and signaling during inflammatory conditions [70]. Notably, CYP1B1 is expressed on cerebral micro-vessels and astrocytes at the BBB interface [71,72], and in conjunction with membrane transporters, may aid in regulating xenobiotic

passage into and out of the brain. CYP2D6 is expressed in neurons and glial cells [68]. CYP2D6 is involved in opioid metabolism and that of many antidepressants, antipsychotics, and detoxification of pesticides [66,73]. Further, in vitro studies have shown that CYPs 3A4, 2A6, 1A1, and 2E1 are expressed in human monocyte-derived-macrophages as well as in astrocytic cell lines [13,49–52]. CYPs 1A1 and 2A6 account for the majority of CYP content in SVGA astrocytes, while CYP2A6 is the predominant isoform in U937 macrophages [13,21]. Monocytes are known to enter the brain from the periphery and differentiate into perivascular macrophages and microglia [74], making their presence in the CNS an important target for CYP activity.

Relatively low levels of brain CYP content may have neurotoxic ramifications. For example, low cerebral 3A4 may make brain tissue more sensitive to pharmaceutical inhibition of psychotropic drug metabolism, as illustrated by ritonavir-mediated inhibition of carbamazepine and consequent ataxia [75]. Moreover, a possible explanation for nicotine-mediated induction of 1A1 and 2A6 observed in astrocytes, but not hepatocytes, may be that hepatic 1A1 and 2A6 are already expressed at maximal levels [13]. Indeed, brain CYPs seem to be particularly sensitive to xenobiotic-mediated induction. Increased expression of CYPs 2E1, 2B6, and 2D6 has been observed in the brain tissue of smokers and alcoholics [14,69,76–78], but changes in hepatic 2B6 and 2D6 are undetectable [78,79]. Our in vitro study showed that CYP2E1 is induced by alcohol exposure in both astrocytes and monocytes via oxidative stress-mediated protein kinase C/c-Jun N-terminal kinase/specificity protein1 (PKC/JNK/SP1) pathways, which eventually causes cellular toxicity. Although evidence suggests CYP2E1-mediated mechanisms of cellular toxicity, including neurotoxicity and contribution to neurological diseases (HAND, PD), it is possible that that cellular CYP content in the CNS is insufficient to meditate these effects. Therefore, it is worth considering the possibility of additional sources of CYP enzymes, which may be transferred to brain cells, when evaluating xenobiotic-induced toxicities and therapeutic efficacy in brain cells.
