*Article* **Farnesoid X Receptor Activation Stimulates Organic Cations Transport in Human Renal Proximal Tubular Cells**

#### **Teerasak Wongwan 1, Varanuj Chatsudthipong 1 and Sunhapas Soodvilai 1,2,\***

Received: 13 July 2020; Accepted: 12 August 2020; Published: 24 August 2020

**Abstract:** Farnesoid X receptor (FXR) is a ligand-activated transcription factor highly expressed in the liver and kidneys. Activation of FXR decreases organic cation transporter (OCT) 1-mediated clearance of organic cation compounds in hepatocytes. The present study investigated FXR regulation of renal clearance of organic cations by OCT2 modulation and multidrug and toxin extrusion proteins (MATEs). The role of FXR in OCT2 and MATEs functions was investigated by monitoring the flux of 3H–MPP+, a substrate of OCT2 and MATEs. FXR agonists chenodeoxycholic acid (CDCA) and GW4064 stimulated OCT2-mediated 3H–MPP+ uptake in human renal proximal tubular cells (RPTEC/TERT1 cells) and OCT2-CHO-K1 cells. The stimulatory effect of CDCA (20 μM) was abolished by an FXR antagonist, Z-guggulsterone, indicating an FXR-dependent mechanism. CDCA increased OCT2 transport activity via an increased maximal transport rate of MPP+. Additionally, 24 h CDCA treatment increased MATEs-mediated 3H-MPP+ uptake. Moreover, CDCA treatment increased the expression of OCT2, MATE1, and MATE2-K mRNA compared with that of the control. OCT2 protein expression was also increased following CDCA treatment. FXR activation stimulates renal OCT2 and MATE1/2-K-mediated cation transports in proximal tubules, demonstrating that FXR plays a role in the regulation of OCT2 and MATEs in renal proximal tubular cells.

**Keywords:** Nuclear receptor; renal excretion; kidney; drug transporters; bile acids

#### **1. Introduction**

The kidney is largely responsible for the elimination of metabolic waste products, therapeutic drugs, and xenobiotics, which contain organic cations (OCs) and anions (OAs) [1]. The secretion of OCs takes place in renal proximal tubules. This process requires the uptake of OCs from the blood into renal proximal tubular cells and subsequent elimination of these compounds into the tubular lumen across the luminal membrane. Three members of the organic cation transporters (OCTs), including OCT1, OCT2, and OCT3, are characterized [2]. Human OCT1 and OCT2 are highly expressed in liver and kidney, respectively, whereas OCT3 is ubiquitously expressed at a low level in multiple tissues [3]. OCs are transported into renal proximal tubular cells via the organic cation transporter (OCT) 2, a predominant OCT expressed in the basolateral membrane of human renal proximal tubular cells [4,5]. OCT2-mediated uptake of OCs is governed by an inside-negative membrane potential [4,6]. After uptake, OCs are then effluxed to the tubular lumen by several apical membrane transporters such as multidrug and toxin extrusion proteins (MATEs). Two MATE isoforms, MATE1 and MATE2-K, are expressed in renal proximal tubular cells [7,8]. Several endogenous compounds and therapeutic cationic drugs are eliminated via renal excretion that have been identified as substrates of both OCT2 and MATEs such as creatinine and metformin [5,9–13].

Altered expression or function of these transporters can affect organic cation drug renal secretion and subsequently alter their pharmacokinetics and efficacies [14,15]. Studies have reported nuclear receptor-mediated regulation of OCT2 and MATEs function. Specifically, the activation of pregnane X receptor (PXR) and androgen receptor increased OCT2 expression [16,17], whereas liver X receptor (LXR) activation decreased OCT2 expression and function [18]. In addition, kidney-specific multidrug and toxin extrusion proteins (MATE2K) expression was up-regulated upon the activation of Nrf2 pathway signaling [19]. Previous studies have shown that farnesoid X receptor (FXR), a ligand-activated transcriptional factor, is highly expressed in liver, kidney, intestine, and adrenal gland tissue [20]. FXR regulates several membrane transporters and channels, including the bile salt export pump (BSEP) [21], multidrug resistance-associated protein 2 (MRP2) [22], organic solute transporter OSTα/β [23,24], aquaporin 2(AQP2) [25], and MATE1 [26].

Previous studies showed that cholestasis resulted in a down-regu lation of OCT1 and impairment of hepatic-mediated OCT1 substrate uptake [27,28]. These processes might be controlled by the activation of FXR by bile acids, such as cholic acid and chenodeoxycholic acid (CDCA). These bile acids have been reported to act as endogenous ligands for FXR [29] and are increased in hepatic disease [30]. Down-regulation of OCT1 regarded as adaptive responses to cholestasis and may serve to diminish the hepatic accumulation of cationic substrate during liver injury [31]. Since OCT1 plays a role in the hepatic uptake-mediated biotransformation and the excretion of endogenous compounds and cationic drugs, decreases in OCT1 function may result in increased cationic plasma concentration. We hypothesize that other cation transporters expressed in the kidney, such as OCT2 and MATEs, may be critically important for cationic substrate elimination in hepatic disease. Here, we investigated the effect of FXR activation on renal OCT2 and MATEs function in renal proximal tubular cells.

#### **2. Results**

#### *2.1. FXR Agonists Stimulate OCT2-Mediated 3H-MPP*+ *Uptake*

To be certain that RPTEC/TERT1 cells are suitable cell model for investigating the role of FXR, we first tested whether the RPTEC/TERT1 cells express FXR by examination protein expression via Western blot analysis. As shown in Figure 1, we confirmed FXR protein expression in RPTEC/TERT1 cells. Relative to untreated cells, mRNA expression of a small heterodimer partner (SHP; a target gene of FRX activation [32,33]) was significantly increased following treatment with 20 μM CDCA for 24 h (Figure 1A,B). Next, we tested the effects of CDCA and GW4064, a potent synthetic agonists of FXR, on OCT2-mediated 3H-MPP+ uptake. As shown in Figure 1C, 24 h incubation with 20 μM CDCA and 5 μM GW4060 significantly stimulated OCT2-mediated 3H-MPP+ cellular uptake. While 20 μM CDCA stimulated uptake after 24 h, an extended incubation time did not lead to any further increase in OCT2-mediated 3H-MPP+ uptake (Figure 1D). In addition, we confirmed the effect of CDCA on OCT2-mediated 3H-MPP+ uptake in CHO-K1 cells expressing OCT2. Specifically, 24 h incubation with 20 and 30 μM CDCA significantly promoted 3H-MPP+ uptake in CHO-K1 cells (Figure 1E).

**Figure 1.** Effect of FXR agonists on OCT2-mediated 3H–MPP+ uptake. (**A**) protein expression of FXR and (**B**) mRNA expression of SHP in RPTEC/TERT1 cells following treating the cells with vehicle or 20 μM CDCA for 24 h; (**C**) effect of 20 μM CDCA and 5 μM GW4064 on 3H-MPP+ uptake; (**D**) Time-response effect of 20 μM CDCA; (**E**) FXR expression and 3H-MPP+ uptake in OCT2-CHO-K1 cells following incubation with 20 μM CDCA for 24 h. Data are expressed as a mean percentage of control (mean ± S.D.) from 3 independent experiments. \*Significantly different from control (*p* < 0.05).

#### *2.2. Stimulatory E*ff*ects of FXR Agonists Require FXR Activation*

To determine whether the CDCA stimulation of 3H-MPP+ uptake is directly caused by FXR activation, we examined how FXR antagonists, Z-guggulsterone and DY268, affect CDCA-induced stimulation of 3H-MPP+ uptake. As shown in Figure 2, exposure to 10 μM Z-guggulsterone or DY268 had no significant effect on 3H-MPP+ uptake. CDCA-mediated uptake stimulation was attenuated by coincubation with Z-guggulsterone or DY268. These data indicate that CDCA uptake stimulation requires FXR activation.

**Figure 2.** Effect of FXR activation on OCT2-mediated 3H–MPP+ uptake. RPTEC/TERT1 cells were treated with vehicle, 20 μM CDCA, FXR antagonists (10 μM Z-guggulsterone (Z-GS) or 10 μM DY268), and CDCA plus FXR antagonist for 24 h. The results are shown as mean ± S.D. of % control form 4 experiments. \*Significantly different from control (*p* < 0.05) and # p < 0.05 compared with CDCA-treated cells.

#### *2.3. Kinetic Study on FXR Activation on OCT2-mediated 3H-MPP*+ *Uptake*

To investigate how FXR activation stimulates 3H-MPP+ uptake, we evaluated the kinetic parameters Kt and Jmax that reflect an affinity and functional membrane expression of OCT2, respectively. As shown in Figure 3, 24 h treatment with 20 μM CDCA in RPTEC/TERT1 cells significantly increased the Jmax from 6.48 ± 1.4 to 12.56 ± 3.1 pmol/min/cm2 with no significant effect on Kt (22.83 ± 5.7 vs 19.75 ± 5.67 μM).

**Figure 3.** Kinetic study of OCT2-mediated 3H-MPP+ uptake in RPTEC/TERT1 cells. RPTEC/TERT1 cells were treated with vehicle or 20 μM CDCA for 24 h. 3H-MPP+ uptake was determined in the presence of unlabeled MPP+ at 0–200 μM. The Jmax, and Kt values are reported as mean ± S.D. (*n* = 3). \*Significantly different from control (*p* < 0.05).

#### *2.4. FXR Activation Increases mRNA and Protein Expression of OCT2*

To determine whether FXR activation affects OCT2 expression, RPTEC/TERT1 cells treated with vehicle or 20 μM CDCA for 24 h were probed for OCT2 mRNA and protein expression. Treatment of RPTEC/TERT1 cells with 20 μM CDCA significantly increased OCT2 mRNA expression compared with vehicle treatment. In addition, 20 μM CDCA treatment led to an increase in OCT2 protein expression as shown in Figure 4.

**Figure 4.** Effect of CDCA on expression of hOCT2. (**A**) mRNA expression of hOCT2 after treating with vehicle or 20 μM CDCA for 24 h. (**B**) Representative blots and the densitometry quantification of hOCT2 expression normalized by β-actin. The data are shown as mean ± S.D. from three independent experiments. \* *p* < 0.05 compared with vehicle-treated group.

#### *2.5. FXR Activation Increases Function and Expression of MATEs*

Regulation of MATEs transport function by FXR activation was determined in RPTEC/TERT1 cells. These transporters function as organic cation/H+ exchangers and are driven by a proton-gradient. Therefore, to test MATEs-mediated 3H–MPP+ uptake, we preincubated RPTEC/TERT1 cells with a K+ based buffer containing ammonium chloride to generate intracellular acidification before transport measurement. Consequently, 20 μM CDCA treatment for 24 h significantly increased MATEs-mediated 3H-MPP+ compared with the vehicle-treated cells. CDCA stimulation was significantly inhibited by Z-guggulsterone and DY268 (Figure 5A). Next, we tested whether the observed correlation between FXR activation and MATEs transport function was a result of MATEs mRNA up-regulation. Using RPTEC/TERT1 cells, MATE1 and MATE2K mRNA expression were analyzed following treatment with 20 μM CDCA for 24 h. CDCA significantly increased both MATE1 and MATE2K mRNA expression (Figure 5B).

**Figure 5.** The effect of FXR activation on MATEs-mediated 3H–MPP+ uptake and MATEs expression in

RPTEC/TERT1 cells. (**A**) MATEs-mediated 3H–MPP+ uptake; treated with vehicle, 20 μM CDCA, FXR antagonists (10 μM Z-guggulsterone (Z-GS) or 10 μM DY268 (DY)), and CDCA plus FXR antagonists. (**B**) mRNA expression of MATE1 and MATE2K. The data are shown as mean ± S.D. (*n* = 3). \* *p* < 0.05 compared with control and # *p* < 0.05 compared with CDCA-treated cells.

#### *2.6. FXR Activation Stimulates Transepithelial Transport of 3H-MPP*+

To determine the relationship between FXR activation and transcellular transport of OCs, we examined the effect of FXR activation on basolateral-apical transport of 3H-MPP+. As such, cell monolayers were incubated with vehicle control, 20 μM CDCA, 10 μM DY268, and 20 μM CDCA plus 10 μM DY268, and transepithelial transport of 3H-MPP+ was measured after 24 h. As shown in Figure 6, transcellular translocation of 3H-MPP+ from the basolateral to the apical chamber was significantly higher in the CDCA-treated cell monolayer compared with the vehicle-treated cells. Importantly, the stimulatory effect of CDCA was abolished by co-treatment with DY268.

**Figure 6.** Effect of CDCA on transcellular transport of 3H-MPP+ in polarized cell monolayer. Polarized RPTEC/TERT1 cell monolayers were treated with vehicle, 20 μM CDCA, 10 μM DY268 (DY), and CDCA plus DY for 24 h. The values of basolateral to apical transport of 3H-MPP+ are expressed as mean ± S.D. of cpm/min/cm2 form 3 experiments. Data from each experiment is obtained from 3 inserts. \* *p* < 0.05 compared with vehicle-treated group and # *p* < 0.05 compared with CDCA-treated cells.

#### *2.7. Pathological Concentration of Bile Acid Stimulates Renal OCT2 and MATEs*

Previous studies have reported an increased concentration of unconjugated bile acids in liver diseases [30]. Therefore, we investigated the correlation between high unconjugated bile acid concentration and the stimulation of renal OCs transport. Cell monolayers were incubated with CDCA at 80 μM for 24 h followed by measurement of OCT2- and MATEs-mediated 3H-MPP+ transport. As shown in Figure 7, treatment a pathological concentration of CDCA significantly stimulated both OCT2- and MATEs-mediated 3H-MPP+ compared with vehicle-treated cells. The stimulatory effect of 80 μM CDCA on 3H-MPP+ uptake was correlated with an increase in mRNA expression of OCT2, MATE1, and MATE2K.

**Figure 7.** Effect of high concentration of CDCA on function and expression of OCT2 and MATEs. RPTEC/TERT1 cells were treated with vehicle or 80 μM CDCA for 24 h followed by measurements of (**A**) OCT2- and MATEs-mediated 3H-MPP+ uptake and (**B**) mRNA expression of OCT2, MATE1, and MATE2K. The data are expressed as mean ± S.D. from 3 independent experiments. \* *p* < 0.05 compared with vehicle-treated group.

#### **3. Discussion**

Cationic transporters play a crucial role in the renal clearance of cationic endogenous and xenobiotic compounds [1,34]. Therefore, the altered expression and/or function of these transporters could affect the total profile excretion of these compounds. Previous reports showed that FXR activation regulates several hepatic transporters in different manners including down-regulation of OCT1 protein expression [27] or up-regulation of MATE1 protein expression [26]. The present study revealed that FXR activation regulates renal OCT2 and MATEs expression and function in the human proximal tubular cell line RPTEC/TERT1. Importantly, RPTEC/TERT1 cells express OCT2, MATE1, and MATE2K and represent an important in vitro model for studying renal transport [35]. Although the expression of FXR is present in the proximal tubular cells, we verified whether RPTEC/TERT1 cell line was suitable as a study model of FXR function. For this study, we initially confirmed that this cell line could be used for studying FXR activation by showing that FXR is expressed and activated by FXR agonist. Of note, we showed that FXR agonists increased OCT2-mediated 3H-MPP+ uptake in RPTEC/TERT1 cells. Importantly, the effect of CDCA on OCT2-mediated 3H-MPP+ uptake was not observed until after 24 h of incubation time; indicating that the FXR agonist has a slow mode of action on OCT2. Moreover, we showed that CDCA modulation of OCT2 is dependent upon FXR activation, as evidenced by our result showing that inhibition of FXR by pharmacological antagonists, guggulsterone and DY268 [36,37], attenuated the CDCA-mediated stimulation. Since RPTEC/TERT1 cells express both OCT2 and OCT3 [35], total 3H-MPP+ uptake into RPTEC/TERT1 cells could be mediated by either. However, we proved here that CDCA also stimulates OCT2 transport function in CHO-K1 cells expressing only OCT2. Taken together, these data imply that FXR activation by CDCA stimulates OCT2-mediated 3H-MPP+ uptake into RPTEC/TERT1 cells. However, we cannot rule out possible stimulatory effects of FXR activation on OCT3 in RPTEC/TERT1 cells. How FXR activation affects OCT3 should be further investigated in cells expressing OCT3 alone.

The stimulatory effect of FXR activation on OCT2 transport might via increase in either the functional number of transporters and/or transporter affinity with its substrate. Using kinetic data, we revealed that CDCA treatment increase in the Jmax of OCT2-mediated transport function. This result is indicative of an increase in the number of transporters at the membrane surface. FXR activation regulates several renal transporters and channel such as the organic solute transporters α and β (OSTα and OSTβ) and AQP2 by increase in mRNA and protein expressions [24,25], we investigated whether increased expression of OCT2 mediated the stimulatory effect of CDCA. We found that CDCA increased OCT2 mRNA and protein expression in RPTEC/TERT1 cells. These results indicate that FXR activation increases 3H-MPP+ uptake into renal proximal tubular cells via the up-regulation of OCT2 mRNA and protein. Although previous studies have demonstrated that FXR modulates transporter and channel gene expression [24,25], we did not explore FXR direct binding and the up-regulation of OCT2 gene expression. FXR direct modulation of OCT2 expression needs to be determined in future studies.

Renal secretion of OCs requires both basolateral uptake and apical efflux. Therefore, we also investigated the correlation of FXR activation with the transporters expressed at apical membrane mediating secretion of OCs including MATE1 and MATE2K [8]. Our data demonstrated that CDCA stimulated the transport function of MATEs. Since FXR antagonists abolish this effect, we showed that the CDCA-mediated stimulation was controlled directly through FXR. The correlative relationship between FXR activation and OCT2/MATEs transport function was confirmed in basolateral to apical experiments. We demonstrated that FXR activation drove the flux of 3H-MPP+ from the basolateral chamber to the apical chamber. These results indicate that FXR activation stimulates OCT2/MATEs-mediated OC secretion in renal proximal tubular cells. Since RPTEC/TERT1 cells express both MATE1 and MATE2K, it is unclear what isoform FXR stimulated. Furthermore, we found that the stimulatory effect of FXR activation on 3H-MPP+ transport correlated well with the up-regulation of MATE1 and MATE2K mRNA. These results provide evidence that FXR activation stimulates OC secretion via up-regulation of both MATE1 and MATE2K.

There is increasing evidence that FXR is a critical regulatory factor in renal physiology and pathophysiology [38]. Previous studies have shown that pathological conditions related to hepatic injuries, such as ischemia/reperfusion and cholestasis, result in an up-regulation of the efflux transporter MATE1 and down-regulation of the uptake transporter OCT1 [27]. Dysregulation of these transporters can lead to the reduction of accumulated cationic compounds in hepatocytes [27]. Altered hepatic function could result in an increased plasma concentration of cations. Pathological conditions in the liver, such as acute hepatitis and obstructive jaundice, significantly increase the total serum concentrations of unconjugated bile acid including cholic acid, deoxycholic acid, and chenodeoxycholic acid (endogenous FXR agonists) up to 100 μM [29,30]. To model the consequences of pathologically high bile acid concentrations, we sought to correlate the modulation of bile acid concentration with renal OCT2, MATE1, and MATE2K expressions and functions. Our results revealed that high CDCA concentrations increase OCT2 and MATEs mRNA expression and modulate their function. These findings correlate with a previous study, which found that renal clearance of OCs in acute hepatic injury was increased due to high protein expression of OCT2 in the renal cortex [39]. Taken together, these data imply that renal FXR activation may be an adaptive response for the clearance of excess plasma OCs during hepatic clearance impairment.

#### **4. Materials and Methods**

#### *4.1. Chemicals*

N-methyl-3H-4-phenylpyridinium acetate (3H-MPP+; 80 Ci/mmol) was purchased from American Radio Labeled Chemical Inc. (St. Louis, MO, USA). DY268 was purchased from Trocris (Thai Can Biotech, Bangkok, Thailand). Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 nutrient mix (1:1), and TRIzol reagent, products of Invitrogen, were purchased from Gibthai (Bangkok, Thailand). iScrip cDNA Synthesis Kit and Luna Universal qPCR mastermix were obtained from Bio-Rad Thailand (Bangkok, Thailand), GW4064 (synthetic FXR agonist). Chenodeoxycholic acid (CDCA), Z-guggulsterone (FXR antagonist), tetrapentylammonium (TPeA), methyl-4-phenylpyridinium (MPP+), and human OCT2 (HPA008567) antibody were purchased from Sigma-Aldrich (Bangkok, Thailand). Antibodies against FXR and β-actin were purchased from Merck Millipore (Bangkok, Thailand). Other chemicals used were of analytical grade from commercial sources.

#### *4.2. Cell Cultures*

RPTEC/TERT1 cells, an immortalized renal proximal tubular cell line expressing several drug transporters [35,40], was obtained from American Type Culture Collection (ATCC) and cultured in a mixture of DMEM and Ham's F-12 (1:1) supplemented with 10 ng/mL human epithelial growth factor, 5 μg/mL insulin, 5 μg/mL human transferrin, 5 ng/mL sodium selenite, 36 ng/mL hydrocortisone, 100 U/mL penicillin, and 100 μg/mL streptomycin. CHO-K1 cells expressing rbOCT2 were kindly gifted from Professor Stephen Wright, University of Arizona. These cells were maintained in Ham's F12 media supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, and 1% G418. All cells were cultured in a humidified incubator with 5% CO2/95% air at 37 ◦C.

#### *4.3. Measurement of OCT2 Transport Function*

OCT2-mediated in 3H-MPP+ uptake in RPTEC/TERT1 cells was measured as previously described [18]. Briefly, RPTEC/TERT1 cell monolayers were washed twice with 1 mL of warm buffer pH 7.40 (NaCl 135 mM, KCl 5 mM, HEPES 13 mM, CaCl2.2H2O 2.5 mM, MgCl2 1.2 mM, MgSO4.7H2O 0.8 mM and D-glucose 28 mM) and incubated for further 15 min. The cell monolayers were incubated with buffer containing 3H-MPP+for 5 min. The transport was stopped by three times washing with ice-cold buffer containing 100 μM unlabeled MPP+. Cells were then lysed by adding 200 μL of 0.4 N NaOH in 10% SDS and left overnight. To neutralize the sample pH, 80 μL of 1 N HCl was added into each well. Accumulation of labeled MPP+ was determined with a liquid scintillation and calculated as mole/min/cm2 of the confluent monolayer surface.

#### *4.4. Measurement of MATEs Transport Function*

Measurement of MATEs-mediated 3H-MPP+ transport in RPTEC/TERT1 cells was performed as described by previous study [41]. Briefly, the cell monolayers were washed twice with 1 mL of warm K+-based buffer (pH 7.4; KCl 130 mM, MgSO4.7H2O 1.2 mM, CaCl2.2H2O 1 mM, K2HPO4 2 mM, HEPES 20 mM, and D-glucose 5 mM) and were incubated for 15 min at 37 ◦C. To manipulate the intracellular acidification, the cell monolayers were further incubated with K+-based buffer containing NH4Cl 30 mM for 20 min at 37 ◦C [42,43]. Then, the cell monolayers were incubated with 200 μl of K+ based-buffer (pH 8.0) containing 3H-MPP+ for 10 min. After incubation, the cell monolayers were washed three times with ice-cold buffer containing unlabeled MPP+ 100 μM to stop transport activity. The cells were lysed by 0.4 N NaOH in 10% SDS and cellular accumulation of 3H-MPP+ was measured and calculated as fmol/min/cm2 of the confluent monolayer surface.

#### *4.5. Basolateral to Apical Transport of 3H-MPP*+

RPTEC/TERT1 cells were cultured in in Transwell 12-well cultures (0.4 μm pore size; Corning Life Science, Corning, NY, USA) for 21 days. Basolateral and apical chambers were filled with 1 and 0.5 mL of media, respectively. Cell monolayer integrity was assessed using transepithelial electrical resistance (TEER). We selected the cell monolayers that achieved TEER values > 100 Ω·cm2. On the day of experiment, the culture medium was withdrawn, and replaced with warm transport buffer and incubated with warm buffer for 30 min at 37 ◦C. Basolateral chamber was added with 3H-MPP+ for 30 min followed by sample collection (0.2 mL) from the apical chamber to determine 3H-MPP+ transepithelial transport. Transporter-mediated 3H-MPP+ transport was calculated by subtraction the total basolateral to apical transport of 3H-MPP+ with the transport of 3H-MPP+ in the presence of TPeA 100 μM, an inhibitor of OCTs.

#### *4.6. Kinetic Analysis of OCT2-mediated 3H-MPP*+ *Uptake*

The evaluation of OCT2 transport kinetics was performed as described previously [18]. RPTEC/TERT1 cell monolayers were incubated with transport buffer containing 3H-MPP+ 10 nM in the presence of various concentrations of unlabeled MPP+. 3H-MPP+ uptake was calculated as

mole/min/cm2 of the confluent monolayer surface. This was followed by the calculation of kinetic parameters including a maximum transport rate of MPP+ (Jmax) and the concentration of unlabeled MPP+ that resulted in half-maximal transport (Kt) using the Michaelis–Menten equation of competitive interaction between labeled and unlabeled MPP+ [44].

#### *4.7. Real-Time PCR*

Total RNA from RPTEC/TERT1 cells was extracted using TRIzol reagent (Invitrogen, Bangkok, Thailand). Synthesis of cDNA was performed using iScript cDNA Synthesis Kit (Bio-Rad, Bangkok, Thailand). A Luna Universal qPCR mastermix was then utilized for PCR amplification (Bio-Rad, Bangkok, Thailand). The primers used in this study are shown in Table 1.

The cycle threshold (CT) values were obtained from ABI Prism 7500 Sequence Detection System (Applied Biosystems (Thailand), Bangkok, Thailand), and the relative expression levels of mRNA were determined by the 2−ΔΔCt method [45].

#### *4.8. Western Blot Analysis*

Proteins of RPTEC/TERT1 cells were separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk for 2 h at room temperature and then blotted with primary antibodies for overnight at 4 ◦C. After that, the membranes were washed four times with Tris-buffered saline (TBST) for 10 min each. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Merck Millipore, Bangkok, Thailand) for 1 h. Proteins were detected and quantified by using an enhanced chemiluminescence (ECL) detection kit (Merck Millipore, Bangkok, Thailand) and the Gel and Graph Digitizing System (Uvitec, Cambridge, UK), respectively.

#### *4.9. Statistical Analysis*

Data are presented as mean and standard deviation (mean ± S.D.). Data of the kinetic study were analyzed by using unpaired student *t*-tests whereas other data were analyzed by using one-way analysis of variance (one-way ANOVA) tests with a post hoc Newman–Keuls test. The significant difference between each group of data was considered when *p* < 0.05.

#### **5. Conclusions**

We have demonstrated that FXR activation stimulates OC secretion in human renal proximal tubular cells. Moreover, the stimulatory effect of FXR on renal OC secretion may be mediated by the increase in OCT2/ MATEs-mediated OC transport. This effect is likely caused by enhanced OCT2 and MATE1/2K expression. Taken together, this study enhances our understanding of the role FXR may play in the regulation of renal OCT2- and MATEs-mediated renal OCs excretion.

**Author Contributions:** T.W. and S.S. conceived the studies and planned the experimental design. T.W. and S.S. performed the experiments analyzed the data. T.W., V.C., and S.S. interpreted the data. T.W. and S.S. wrote manuscript. V.C. and S.S. edited and proved the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project has been supported by the Thailand Research Funds and Mahidol University (grant no. RSA6280082 to S.S.) and the Royal Golden Jubilee (RGJ; grant no. PHD/0238/2553 to T.W.).

**Acknowledgments:** We would like to thank Professor Stephen Wright, University of Arizona for providing OCT2-CHO-K1 cells.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


*3.4. Confirmatory Factor Analysis of CBB Model and CBB-R Model*

Table 4 analyzes the fit of the various models of the questionnaire by the original CBB model, the unidimensional CBB model, the proposed four-factor CBB model, and the revision of that model first proposed (CBB-R). The original model and the unidimensional model show values that are not very adequate. The proposed four-factor CBB model, which corresponds to what was found in the exploratory analysis, is better, but while it showed good fit indices, the advisable respecifications were made considering theoretical and statistical criteria (modification indices, errors of estimation, standardized errors of measurement), which led to elimination of items 2, 16, 3, 13, 17, and 11. The revised model showed much better fit with the calibration sample. The difference between the AIC default model value (248.497) and the AIC saturated model value (240.000) is also very small, showing that this is probably the best of the models according to the Akaike model selection criteria.

Fit indices for the proposed CBB-R model with the validation sample (*n* = 635) are shown in Figure 2. Confirmatory factor analysis for the proposed model was done, taking the following fit indices as measures: *χ*2/*gl =* 2.241, CFI = 0.961, TLI = 0.951, RMSEA = 0.044 (0.036–0.053).

**Table 4.** Fit indices for the models proposed (calibration sample; *n* = 605).

Note: CFI, comparative fit index; TLI, Tucker–Lewis index; RMSEA, root mean square error of approximation; CI, confidence interval; *df*, degrees of freedom; Est., estimation; Bel., below; Abv., above. CBB: CBB-R (Revised CBB Model).

The reliability of the model was analyzed using Cronbach's alpha, where α = 0.89 for the total sample; for factor 1 (job dissatisfaction), comprising four items, α = 0.697; for factor 2 (social climate), made up of three items, α = 0.666; for factor 3 (personal impact), made up of four items, α = 0.808; and for factor 4 (motivational exhaustion), comprising four items, α = 0.529. Furthermore, the data found by split halves also showed both equal-length (Spearman–Brown coefficient = 0.818) and unequal-length (Spearman–Brown coefficient = 0.819) consistency of the scales.

**Figure 2.** Proposed CBB-R model (validation sample *n* = 635). Note: F1: job dissatisfaction; F2: social climate; F3: personal impact; F4: motivational exhaustion.

Table 5 shows the values for all six models. It can be seen how ΔCFI is over 0.01 for models 3 and 4, accepting the configural and metric invariance. Specifically, ΔCFI between model 1 (configural and metric base model) and models 3 and 4 is 0.024, so scalar and strict invariance cannot be accepted. In the analysis of variance by gender, in all cases ΔCFI is under 0.01, so the configural, metric, scalar, and strict invariances are accepted.

**Table 5.** Multigroup analysis of variance by type of contract (permanent/temporary) and gender (male/female).

Nota FS = factor saturation; Int = intercepts; Err = error.


*Article* **Inspection Interval Optimization for Aircraft Composite Tail Wing Structure Using Numerical-Analysis-Based Approach**

**Salman Khalid 1, Hee-Seong Kim 2, Heung Soo Kim 1,\* and Joo-Ho Choi 2**

**Abstract:** Recently, there has been a tremendous increase in the use of fiber-reinforced composite (FRCP) in the aviation and aerospace industries due to its superior properties of high strength, stiffness, and low weight. The most important feature of implementing composite materials in aviation is their behavior under dynamic loads and resistance to fatigue. To predict the life of composite structures and optimize the inspection interval, it is essential to predict the damage behavior of composites. In this study, a model of fatigue delamination damage of composite specimens was first constructed using a finite element analysis (FEA)-based approach. The FEA modeling was verified through comparison with experimental specimen data, and the verified FEA model was applied to the composite material aircraft tail wing structure. In this case, a Monte Carlo simulation (MCS) was performed by building a response surface model while considering the uncertainty of the mechanical parameters. Through this process, the risk as a function of flight time could be quantitatively evaluated, and the inspection interval was optimized by selecting the combination with the lowest number of repeated inspections that met the permitted risk criteria.

**Keywords:** fiber-reinforced composites; finite element analysis; delamination; inspection interval; aircraft tail wing structure

**MSC:** 65-04


**6.1 Classification of AMR**

pAMR 0 negative for pathologic AMR: histopathologic and immunologic studies are both negatives [3, 6];

pAMR 1 (H+)—histopathologic AMR alone: histopathologic findings present and immunopathologic findings negative;

pAMR 1 (I+)—immunopathologic AMR alone: immunopathologic AMR alone: histopathologic findings negative and immunopathologic findings positive, that is, CD68+ and/or C4d + for IHC and C4d + with or without C3d + for IF.

pAMR 2—pathologic AMR: histopathologic and immunopathologic findings are both present.

pAMR 3 severe pathologic AMR: interstitial hemorrhage, capillary fragmentation, mixed inflammatory infiltrates, endothelial cell pyknosis, and/or karyorrhexis and marked edema and immunopathologic findings are present.


*Crude Distillation Unit (CDU) DOI: http://dx.doi.org/10.5772/intechopen.90394*

between the initial boiling point of the components. As a result, the oil is divided into fractions up to fuel oil and tar and even base oil. The distillation of oil can be carried out [34]:

The single evaporation of oil is a one-step separation technique. The single evaporation process involves heating the oil to increase the temperature and enthalpy to the true boiling point (TBP) of the vapor-liquid mixture. The process of multiple evaporations is a sequence of single evaporation with a gradual increase in the heating temperature. Distillation by gradual evaporation is a slight change in the oil's state with every single evaporation. The main devices in which the distillation of oil are distillation columns, reboilers, furnaces, etc. [1].

The difficulty of separating crude oils is mainly determined by the volatility of the key components or by the difference in the boiling point of the key components [34]. The closer the relative volatility is to one per mole, the more plates or plates are needed and more irrigation is needed to achieve the same purity of distillate and residue. For key components, two adjacent components are taken for key components, one belonging to the distillate, the other to the residual product. The relative volatility of these adjacent components and their proportion in crude oil are the main criterion determining the difficulty of sufficiently clear separation from crude oil [34]. More the tangent of the angle of inclination of the TBP increase, the easier the separation conditions are. The wider the crude oil TBP intervals, more the distillate and the residue obtained during the process are clean. As we all know, crude oil from the bowels of the Earth contains salt as dissolved salt in a tiny drop of water that forms water into an emulsion [1, 9]. This water cannot be completely separated by gravity or mechanical means. The deep separation of water from the emulsion occurs with electrostatic at the plant, before the distillation process [20].

After desalting process, the crude oil is heated in heat exchangers or reboilers (preheating of crude oil). Of course, preheating is not sufficient, since the oil must be partially evaporated to the extent that all products except atmospheric residues must be in the vapor phase when the oil enters the atmospheric column. Thus, the furnace is required to raise the temperature between 330 and 385°C depending on the components of oil [35]. The partially evaporated crude oil is transferred to the flash zone column located at a point below the distillation column and above what is called the stripping section. The main distillation column is generally up to 50 m with a 30–50 valve [34]. The size of the column is determined by the number of plates and the amount of steam. Besides, the amount of steam is determined by the content of crude oil in volatile elements or compounds. As a result, the rising steam in huge amounts and at top flow rates, requires a large diameter column above the flash zone [1, 34]. At the bottom of the section, water vapor is injected into the column to remove the atmospheric residue of any light hydrocarbon and reduce the partial pressure of hydrocarbon vapors in the flash zone [20]. This causes the true boiling point of hydrocarbons to decrease and causing more hydrocarbons to boil and raise the column to eventually thicken and removed as lateral flows. As hot vapors grow from the flash zone, they ascend into the column through the plates to the upper zone of the column [36]. A portion part of the light fraction of naphtha or gasoline returns to the column in the form of reflux. This reflux allows controlling

the quality of the distillate and the pressure in the column. The main products of atmospheric petroleum distillation are [33]:

The distillation process is carried out in columns with plates inside and it is as follows: hot vapors, rising on the column, in contact with the cooler liquid (reflux) flowing down. There is a cooling of the vapors, hence condensation; a transition from vapors to liquid. This is the process of mass transfer and heat exchange; the contact efficiency is provided by the plates. Most of the side currents (light fractions) pass through the striping section, where they are again dispersed to control their starting and ending boiling point, while the liquid is heated, the low boiling components evaporate [36]. The temperature and pressure have a great influence on the processes (movement of fluids) due to the plates inside the column [32]. The distillation plates are designed to create close contact between Steam and liquid during the grinding process. Mainly apply grid, groove, cap, s-shape, valve, and other types of plates. The design of the plates, besides the close contact between steam, and liquid must ensure sufficient performance of the column, have a low hydraulic resistance to the flow of steam. For example, the movement of vapors in distillation columns depends on the types of plates shown in **Figures 9** and **10** [32]. The materials used in the design are also of great importance because they influence the metal capacity of structures, ease of assembly, and cleaning.

The quality of distillation depends on the number of plates in the column and the

In oil refining, the first oil separation process is very often operated with complex columns or main columns [20]. The oil is sent to the atmospheric distillation column and after the desalination process. The oil is supplied to the feeding area in the form of steam, liquid, or vapor-liquid mixture. This area is called evaporative. Above the entrance (feeding zone) to the oil is the separation zone of the column, and below-the heating zone. In the evaporation zone of the column, there is single evaporation of the oil heated long before in the furnace or the heat exchanger. The purpose of atmospheric distillation is the primary separation of various "Fractions" of hydrocarbons: fuel gases, liquefied petroleum gas, naphtha, kerosene, diesel fuel, and from the bottom of the distillation; the atmospheric residue (heavy hydrocarbon residue). The residues from the atmospheric distillation column or atmospheric residues are sent to the vacuum distillation column. The atmospheric residues in the vacuum column (under the vacuum condition) and temperatures above 400°C give

In oil refining, a fairly large number of plate types are widespread; new types are being developed. This situation is explained by the desire to develop more productive and economic plates than those used previously. The choice of the types of plate depends on a load of steam and liquid, the physical properties of steam and liquid [34]. In order to ensure optimal conditions for the operation of columns in various technological processes, it is necessary to use plates of different designs [32]. In atmospheric columns, steam and liquid charges are moderate. While in vacuum columns, large vapor loads and small liquid loads are observed, that is low volumetric expenditures of liquid and high linear

velocities of vapors. Thus, for some varieties of columns, several types of plates are

amount of irrigation. The speed of movement of vapors in the column and the distance between the plates is great significance. The design of distillation plates is important in the column's performance. Thus, under practical conditions, the heat exchange between steam and liquid on the plate does not reach the equilibrium state, so the concept of plate efficiency (energy conversion efficiency) was introduced. It depends on the design and operating conditions and usually varies between 0.4 and 0.8 [32]. The distillation column comprises three (3) zones: the separation zone, the crude supply (feeding zone), and the heating zone. The feeding zone is generally below the separation zone. However, in some columns like the stripping columns, the feeding zone is located in the upper area of the column. Also, we can say that the separation zone in this type of column is non-existent. In short,

this type of column is a column stabilization.

*The movement of steam and liquid flows on the cap plates.*

*Crude Distillation Unit (CDU)*

*DOI: http://dx.doi.org/10.5772/intechopen.90394*

**Figure 10.**

new fractions (heavy gasoil) [1, 32].

**125**

**2.4 The plates for distillation columns of oil plants**

#### **2.3 Description of the distillation column and its operation**

The distillation is designed to separate gas and liquid mixtures comprising two or more components or fractions. We can classify distillation columns according to various characteristics [32, 34]:

**Figure 9.** *The types of plate in the column: (a) cap, (b) grid, (C) valve.*

**Figure 10.** *The movement of steam and liquid flows on the cap plates.*

The quality of distillation depends on the number of plates in the column and the amount of irrigation. The speed of movement of vapors in the column and the distance between the plates is great significance. The design of distillation plates is important in the column's performance. Thus, under practical conditions, the heat exchange between steam and liquid on the plate does not reach the equilibrium state, so the concept of plate efficiency (energy conversion efficiency) was introduced. It depends on the design and operating conditions and usually varies between 0.4 and 0.8 [32]. The distillation column comprises three (3) zones: the separation zone, the crude supply (feeding zone), and the heating zone. The feeding zone is generally below the separation zone. However, in some columns like the stripping columns, the feeding zone is located in the upper area of the column. Also, we can say that the separation zone in this type of column is non-existent. In short, this type of column is a column stabilization.

In oil refining, the first oil separation process is very often operated with complex columns or main columns [20]. The oil is sent to the atmospheric distillation column and after the desalination process. The oil is supplied to the feeding area in the form of steam, liquid, or vapor-liquid mixture. This area is called evaporative. Above the entrance (feeding zone) to the oil is the separation zone of the column, and below-the heating zone. In the evaporation zone of the column, there is single evaporation of the oil heated long before in the furnace or the heat exchanger. The purpose of atmospheric distillation is the primary separation of various "Fractions" of hydrocarbons: fuel gases, liquefied petroleum gas, naphtha, kerosene, diesel fuel, and from the bottom of the distillation; the atmospheric residue (heavy hydrocarbon residue). The residues from the atmospheric distillation column or atmospheric residues are sent to the vacuum distillation column. The atmospheric residues in the vacuum column (under the vacuum condition) and temperatures above 400°C give new fractions (heavy gasoil) [1, 32].

#### **2.4 The plates for distillation columns of oil plants**

In oil refining, a fairly large number of plate types are widespread; new types are being developed. This situation is explained by the desire to develop more productive and economic plates than those used previously. The choice of the types of plate depends on a load of steam and liquid, the physical properties of steam and liquid [34]. In order to ensure optimal conditions for the operation of columns in various technological processes, it is necessary to use plates of different designs [32]. In atmospheric columns, steam and liquid charges are moderate. While in vacuum columns, large vapor loads and small liquid loads are observed, that is low volumetric expenditures of liquid and high linear velocities of vapors. Thus, for some varieties of columns, several types of plates are recommended, depending on the particular conditions of operation and use of these columns. These operating conditions of the column provide a comparative assessment of the different types of plates under these operating conditions as shown in **Table 3**. The comparison is carried out on the following characteristics and indicators [34]:

*2.4.3 The valve plates*

*Crude Distillation Unit (CDU)*

*DOI: http://dx.doi.org/10.5772/intechopen.90394*

of steam [30, 36].

*2.4.4 The jet plates*

data [25]:

t1 = 250°C;

t2 = 380°C;

**Table 4**.

**127**

bar at the outlet of the liquid from the plate [32].

*2.5.1 Determination of the yield (material balance) of the furnace*

• the pressure at the exit of the furnace, p = 2 bars;

Fn emol ð Þ¼ X

top product's distillate, with the Eq. (35).

**blocks or units like the furnace**

The valve plates are mainly used in CDU or AVDU and gas plants. The dominant element of the plate is a valve of round or rectangular shape that closes the holes at the base of the plate of appropriate shape. Structurally, the valve is made so that it cannot be raised to a certain height. The arrangement of the valves of round shape on the plane of the plate is the same as the plugs on the cap plate. The valve plate is used that rises with increasing steam velocity and then decreases as the stream velocity decreases, which stops the liquid from flowing. The valve can be round or rectangular, with or without a frame. The disk rises with an increase in the velocity

The jet plate consists of a flat sheet, on which there are offset slots made in the form of a tongue with the cut-out part bent upwards. All slots are bent at a side angle. The plate has a drainage device of the usual design, but does not have a drain

**2.5 Examples of material balances of crude distillation unit and with its major**

The role of the furnace is to heat the oil to a temperature in the range of 330–385°C in order to achieve a vapor-liquid balance before the distillation column [1, 7, 25, 29]. The most popular furnace in the Russian Federation is the tube furnace, as shown in **Figure 11**. The tube furnace is a unit designed to heat and force chemical processes inside the chamber, which are achieved by using the heat generated when fuel is burned. To calculate the heat balance of the atmospheric column, it is necessary to determine the heat of the furnace by the vapor phase and the liquid phase, therefore determine the material balance of the furnace. For example, the furnace material balance we used Bonga crude and the following

• the temperature at the entrance of raw materials into the convection chamber

• the data of the molar and mass composition of desalted crude are presented in

We use the method of gradual approximation to calculate the percentage of the

*Cmoli*

1 þ *emol* � ð Þ *Ki* � 1 (35)

*i*

• the temperature at the outlet of raw materials in the convection chamber

As already noted above, there is a multitude of types of plates. Then we will talk about the most frequent plates in oil plants.

### *2.4.1 The lattice or grid plates*

The plates with holes, usually have holes on 3/16 inch to 1 inch. Pairs come out of the holes to get a multi-hole effect. The Steam or vapor velocity prevents liquid from passing through the holes. The number and size of the hole are based on the fall of steam in the column. The fluid flow is transported down the tower using triggers, a dam, and a bypass device on the side of the plate. The Minimum capacity sieve tray capacity is about 70% [30].


**About the Editors**

### **Denis Benasciutti**

Denis Benasciutti is Associate Professor of Machine Design at the University of Ferrara, Italy. In 2001 he graduated cum laude in Materials Engineering at the University of Ferrara, where, in 2005, he also received his Ph.D. In 2006, he earned a Master in Welding Engineering and the diploma as European/International Welding Engineer (EWE/IWE). From 2006 to 2015, he was Assistant Professor at the University of Udine, Italy. His research interests involve the structural integrity of mechanical components subjected to stochastic uniaxial and multiaxial loadings (with special focus on frequency-domain spectral methods) and the cyclic plasticity and low-cycle fatigue behaviour of traditional and additive manufactured materials. He published over 80 scientific articles in peer reviewed journals, on the topics of metal fatigue and structural durability, finite element method, "energy harvesting", cyclic plasticity of metals.

### **Mark T. Whittaker**

Professor Mark Whittaker is the Director of the Institute of Structural Materials at Swansea University, and has a background in high-temperature materials. Following his Ph.D. in 2003, he undertook postdoctoral research mainly based around fatigue lifing of titanium alloys, before accepting an RCUK Research Fellowship in 2007, leading to promotion to Senior Lecturer in 2012, Associate Professor in 2015, and Professor in 2019.

His research interests over this time have extended to high temperature lifing of materials with a particular focus on material behaviour under creep and thermo-mechanical fatigue conditions. Collaboratively working with Professor Brian Wilshire for 5 years enabled the further development of the Wilshire Equations, with the aim to relate concepts from the equation, such as activation energy to micromechanical behaviour, an activity which is still being pursued currently.

Prof Whittaker has worked extensively with industrial partners including Rolls-Royce, TIMET, Pratt & Witney, and Forged Solutions. He also collaborates with national and international research partners and has published over 60 papers in peer reviewed journals, also making invited presentations at a number of international conferences.

### **Turan Dirlik**

Turan Dirlik is the managing director of Dirlik Controls Ltd, UK, supplying bespoke materials testing software to research institutions. Turan's Ph.D. thesis focused on developing a fatigue life assessment technique, using the rainflow cycle counting process. This method is now commonly known as the *Dirlik Formula*. His current work includes fatigue crack propagation and thermo-mechanical fatigue.


**3.2 Himalayan TZ**

The Himalayan TZ marks the continent-continent collision zone with the ongoing thrusting of the Tibetan plate over the Indian plate. This zone, therefore, is highly vulnerable to earthquake generation. Epicentres of several disastrous earthquakes are located in this zone. The northward motion of the Indian plate is constrained by this collision front which is building up strain in this zone and also back thrust that is responsible for the compressive stress experienced in the sub-continent.


**Author details**

Another potential health and safety concern with the general use of sunscreens is the potential of vitamin D deficiency. Appropriate vitamin D synthesis necessitates certain levels of UVB exposure and the American Academy of Dermatology has recently revised its position on sunscreen effects on adequate vitamin levels. They note several populations are at higher risk for vitamin D deficiency including the elderly, darker skinned those who live in areas of low sunlight exposure, the obese, and the photosensitive. It is generally recommended that individuals obtain 1000 international units of vitamin D daily and some may have to supplement

Unlike other organic sunscreens, the inert nature of titanium dioxide sunscreens makes it one of the least likely compounds to cause irritation and sensitization of the skin. In fact, because of its chemical inertness and lack of photoreactivity, it is often the preferred sunscreen for individuals with sunscreen allergies and children [3]. However, in recent years an interesting interaction between gold allergic patients and titanium dioxide has been proposed and studied. In 2005, Nedorost and Wagman hypothesized that patients with facial and eyelid dermatitis may be experiencing reactions due to an interaction between their gold jewelry (earrings, bracelets, etc.) and the titanium dioxide in their cosmetic products. Mechanistically, titanium dioxide was thought to adsorb gold particulate matter which then caused contact dermatitis in the locations of makeup application. Their results demonstrated a subgroup of

benefiting from gold jewelry avoidance, lending credence to the interaction theory [22]. In 2015, Danesh and Murase published a clinical pearl in the Journal of the American Academy of Dermatology regarding a trial of gold avoidance in patients presenting with eyelid allergic contact dermatitis. They noted that the North American Contact Dermatitis Group implicated gold as the most common allergen to cause eyelid dermatitis most likely through a mechanism in which eyelid cosmetics containing titanium dioxide particles adsorbed gold particles

The American Academy of Dermatology has set forth guidelines for appropriate sunscreen use and sun protection. They recommend using sunscreen with broad-spectrum properties (protection against both UVA and UVB radiation) daily. These include sunscreens with sun protection factors greater than 30. Sunscreen should be applied 15–30 min prior to sun exposure and be reapplied as needed when environmental factors (swimming and sweating) cause

In conclusion, titanium dioxide sunscreens are effective agents against UV radiation. They also have an advantage as they protect against multiple wavelengths of energy

**7. General recommendations on appropriate sunscreen use**

containing makeup on areas then affected by contact dermatitis,

this through oral formulations [3].

68 Application of Titanium Dioxide

individuals who used TiO2

from elsewhere on the body [23].

sunscreen removal [3].

**8. Conclusions**

Megha Trivedi1,2\* and Jenny Murase2,3

\*Address all correspondence to<EMAIL_ADDRESS>
1 University of Michigan Medical School, Ann Arbor, MI, USA

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