**1. Introduction**

Cytochrome P450s (CYPs) are a superfamily of enzymes located either in the inner membrane of mitochondria or in the endoplasmic reticulum membrane of eukaryotic cells. There are 57 CYP proteins encoded in the human genome, which are responsible for the metabolism of numerous endogenous and exogenous compounds [1–3]. CYPs mainly oxidize these compounds to generate more hydrophilic metabolites, enhancing their excretion outside the body and thus playing a major role in the detoxification of toxic chemicals [1]. Generally, CYP families 1, 2, and 3 include major xenobiotic-metabolizing enzymes responsible for their major roles in pharmacogenomics risk, while CYP4 enzymes are involved in the metabolism of fatty acids, with their close links to genetic disease risk. Fatty acid metabolism by CYP4 enzymes is responsible for the elimination of excess free fatty acids from the body, as well as for the synthesis of proper levels of bioactive fatty acid molecules [4]. The present review focused on the CYP4 family of enzymes in terms of their functional roles, genetic variations, and influences on human diseases.

#### **2. Classification and Tissue Distribution of the CYP4 Family**

Although there are more than 11 subfamilies of CYP4 in different species, only 6 subfamilies of *CYP4* genes have been reported in humans. The human CYP4 subfamilies are CYP4A, B, F, V, X, and Z [2]. Seven *CYP4F* isoforms, *CYP4F2, CYP4F3A, CYP4F3B, CYP4F8, CYP4F11, CYP4F12*, and *CYP4F22*, are clustered on chromosome 19 and are encoded by six genes. *CYP4A* isoforms include *CYP4A11* and *CYP4A22* on chromosome 1 [3], and the remaining *CYP4* subfamily genes are *CYP4B1*, *CYP4V2*, *CYP4X1*, and *CYP4Z1* [4]. The major sites of CYP4A11 expression are the liver and kidney [5]. However, Jarrar et al. found that CYP4A11 protein was also highly expressed in human platelets to a similar level as in the human liver [6]. CYP4A22 expression has been reported in the human liver at very low levels, with poor enzyme activity compared to that of CYP4A11 [7]. Among the seven *CYP4F* genes, *CYP4F2*, *CYP4F3B*, *CYP4F11*, and *CYP4F12* are mainly expressed in the liver and kidney [8–12]. However, their relative contributions to the total amount of CYP4 enzymes in tissues are difficult to determine, as the high structural homology of these four enzymes has hampered the production of specific antibodies for the detection of each enzyme. In addition to the lack of specific antibodies, genetic polymorphisms and differing profiles of up- and downregulation among CYP4 enzymes have further complicated determination of the intrinsic amount of each enzyme in tissues. Currently, mass spectrometry is used to detect target proteins through measurement of specific peptides of the target protein [13,14]. The total amount of CYP4F protein in human liver was estimated as 18–128 pmol/mg liver microsomal protein [15]. One of the most abundant P450s, CYP3A4, was estimated at 64 pmol/mg liver microsomal protein [13], indicating that the contribution of CYP4F to the total P450 level is large. CYP4F3A is expressed in neutrophils and plays a major role in inflammation [16]. CYP4F8 is expressed in the prostate and seminal vesicles [17]. CYP4F22 is expressed in human skin and plays a major role in formation of the skin lipid barrier [18]. CYP4V2 is widely expressed in the liver and ophthalmic tissues and CYP4V2 defect has been linked to ophthalmic diseases, such as Bietti's crystalline dystrophy [19]. CYP4B1 is expressed mainly in the lung and bladder tissues, and in smaller amounts in the liver [20]. CYP4X1 is expressed in the brain and bronchial airways [21], while CYP4Z1 is expressed in mammary tissue; these proteins are also overexpressed in cancer compared to normal cells [22]. Expression levels of CYP4 proteins are summarized in Table 1.



#### **3. Role of the CYP4 Family in the Metabolism of Endogenous Compounds**

The CYP4 family plays a major role in the metabolism of fatty acids, in most cases through oxidation of fatty acids and subsequent catalysis in the mitochondria to produce cellular energy. CYP4B metabolizes short-chain fatty acids (approximately 7 to 10 carbon fatty acids) [20], while CYP4A and CYP4V metabolize intermediate-chain fatty acids (C10 to 16) [19] and CYP4F catalyzes long-chain fatty acids (C16 to 26), such as prostanoids [43]. Decreasing the expression levels of the CYP4 family was associated with accumulation of fats in tissues such as the liver [44]. Therefore, decreased levels of CYP4 family proteins reduce the capacity for fat removal from tissues. Jarrar et al. [44] found that non-steroidal anti-inflammatory drugs caused fatty livers in treated mice, which were associated with significant downregulation of mouse *cyp4a12* gene expression in liver tissues. CYP4F2, 4F3B, 4A11, and 4V2 were found to oxidize arachidonic acid through ω-hydroxylation to 20-hydroxyeicosatetraenoic acid (20-HETE) [6,9,45,46], which is a vasoconstrictor and activator of platelet aggregation [47]. Several studies have reported that CYP4F and CYP4A are overexpressed in cardiovascular diseases, wherein they are correlated with 20-HETE production [48–50]. In addition, doxorubicin-induced cardiotoxicity was associated with increased 20-HETE production due to increased mRNA expression of rat CYP4A and CYP4F enzymes [51]. CYP4A11 and 4V2 oxidize saturated fatty acids such as lauric acid [23,41,52]. In addition to the metabolism of arachidonic acid and omega-3 polyunsaturated fatty acids, CYP4F2 has been reported to ω-hydroxylate leukotriene (LTA) 4 [53]. CYP4F3A in white blood cells catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, which is an important regulatory step of the inflammatory response [54]. Instead of ω-hydroxylation, CYP4F8 has been reported to hydroxylate prostaglandin (PG) E2 at position 19 [17]. Although epoxyeicosatrienoic acids (EETs) are synthesized by the CYP2C subfamily [55], they can be further ω-hydroxylated by CYP4 enzymes to 20-hydroxyepoxyeicosatrienoic acids (HEETs) [56].
