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From Genotype to Phenotype: Cytochrome P450 2D6-Mediated Drug Clearance in Humans Jie Gao, Xin Tian, Jun Zhou, Ming-Zhu Cui, Hai-Feng Zhang, Na Gao, Qiang Wen, and Hai-Ling Qiao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00920 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

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Molecular Pharmaceutics

From Genotype to Phenotype: Cytochrome P450 2D6-Mediated Drug Clearance in Humans

Jie Gao1, Xin Tian1, Jun Zhou1, Ming-Zhu Cui1, Hai-Feng Zhang1, Na Gao1, Qiang Wen1 and Hai-Ling Qiao1,*

1

Institute of Clinical Pharmacology, Zhengzhou University, Zhengzhou, China.

Running Title: Genotype-phenotype relationship for CYP2D6

* To whom the correspondence should be addressed. Hai-Ling Qiao, Institute of Clinical Pharmacology, Zhengzhou University No. 40, Daxue Road, Zhengzhou, Henan 450052, China Email: [email protected]. Tel: +86-0371-66658363. Fax: +86-0371-66658363

ACS Paragon Plus Environment


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Table of Abstract Graphic


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ABSTRACT How genotypic variation results in phenotypic differences is still a challenge for biology. In the field of drug metabolism the means by which specific cytochrome P4502D6 (CYP2D6) genotypes yield different phenotypes at various levels (molecular, cellular, and organismal) is an important question, as differences in CYP2D6 activity can contribute to adverse drug reactions. Herein, the genotype of CYP2D6 was determined along with the absolute content of CYP2D6 and microsomal protein per gram of liver in human liver microsomes, the molecular, cellular (microsomal, tissue, organ), and organismal phenotype of CYP2D6 determined; the effect of genotype on each phenotype of CYP2D6-mediated dextromethorphan clearance (CL) was delineated, and the overall genotype-phenotype relationship for CYP2D6 was charted. We demonstrate that changes in the cellular and organismal CL phenotypes are markedly greater than changes seen at the molecular level. With individuals carrying the 1661CC polymorphism, for example, the most noticeable change took place in organ CL phenotype (4.17-fold), followed by tissue (3.75-fold), organism (3.69-fold), microsomal (3.09-fold), and molecular (1.66-fold) phenotypes. In addition, the biggest intra-genotype individual coefficient of variation in organismal phenotype was observed in the 1661GG individuals, reaching 104.5%, followed by that of 100TT, 100CT, 1661GC, 100CC, and 1661CC polymorphisms (102.7%, 62.4%, 53.5%, 49.7%, and 44.8%, respectively). Our study has allowed us to chart the genotype-phenotype relationship for CYP2D6 from the molecular to the organismal level as well as allowed us to determine intra-genotype individual variation in phenotype with each genotype.

KEYWORDS: CYP2D6, dextromethorphan, drug clearance, genotype-phenotype



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relationship, intra-genotype individual variation

Abbreviations: BW, body weight; CL, clearance; CLH, clearance at the whole organism (in vivo) level; CLL, clearance at the liver level; CLLT, clearance at the liver tissue level; CLM, clearance at the microsomal level; CO, cardiac output; CLP, clearance at the protein level; CV, coefficient of variation; fu,p, plasma unbound fraction, LW, liver weight; MPPGL, microsomal protein per gram of liver; QH, hepatic blood flow; RB, ratio of the drug concentration in blood to plasma; SNP, single nucleotide polymorphisms.

1. INTRODUCTION Heredity information is carried by the gene, but the expression of this information is manifested at the protein level. Changes in genetic information alter the genotype, whereas changes in protein expression or activity alter the phenotype, which can be evidenced at the molecular, cellular, and organismal level. The molecular phenotype is defined as the expression and activity or functionality of the encoded protein; the cellular phenotype reflects this functionality at the sub-cellular, cellular, and organ levels; and the organismal phenotype integrates these individual effects across the whole organism. For drug metabolizing enzymes, molecular phenotype is drug clearance at the protein level (CLP); cellular phenotype is drug clearance at the microsomal (CLM), liver tissue (CLLT), and liver (CLL) levels; and the organismal phenotype is drug clearance at the whole organism (in vivo) (CLH) level. As Prof. Trudy F. C. Mackay said in Nat Rev Genet, “A major challenge of contemporary biology is to understand how naturally occurring genetic variation causes phenotypic variation in quantitative traits.”

1

Unfortunately, for most proteins


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there have been no reports that revealed how specific genotypes yield different phenotypes at various levels. So the relationship between genotype and phenotype remains an important issue. With cytochrome P450 (CYP450) enzymes there are many in vitro drug metabolism

2-4

and pharmacokinetics in vivo studies

5, 6

that investigate the great

variation in drug efficacy and toxicity between different individuals. In vitro drug metabolism studies (e.g. human liver microsomes, hepatocytes, etc.) mainly focus on the effect of CYP genetic polymorphisms on the enzyme’s activity

7-10

. In vivo

pharmacokinetics studies mainly focus on the effect of CYP genetic polymorphisms 11-14

on the pharmacokinetic data (drug clearance)

. There has been a lack of studies

which integrate the effect of genotype on these phenotypes at all levels (from enzyme activity to organismal clearance). In addition, independent results of in vitro and in vivo studies often are inconsistent; for example, in vitro studies have found that the CYP2D6*4 polymorphism is associated with a lack of enzyme activity

15-17

, while in vivo studies have found that

CYP2D6*4 is not associated with a change in tamoxifen response in postmenopausal patients with breast cancer, despite the fact that tamoxifen is a substrate for CYP2D6 18-20

. We previously found that CYP2A6, CYP2B6, and CYP3A4/5 did not affect,

while CYP2C9 and CYP2D6 increased drug clearance in vitro in patients with hepatocellular carcinoma, while CYP2A6, CYP2B6, and CYP3A4/5 decreased, while CYP2C9 and CYP2D6 did not change drug clearance in vivo

21

. Hence, there is a

clear need to understand how changes in enzyme activity at the subcellular level (resulting from genetic polymorphisms) affect whole organism drug clearance. CYP2D6 has been widely studied owing to its numerous polymorphisms that affect metabolic activity, and its role in the metabolism of many classes of drugs, including



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selective serotonin reuptake inhibitors, antidepressants, β-blockers, anti-arrhythmics, and neuroleptics

22, 23

. The CYP2D6 gene, located on chromosome 22q13.1, has one

of the highest numbers of genetic polymorphisms, with over 105 allelic variants 24, 25. It has been reported that many variations of CYP2D6 generate absent or non-functional proteins, or result in increased protein expression, yielding wide inter-individual and inter-ethnic variabilities in CYP2D6 activity

22

. Therefore, the

CYP2D6 genotype relationship to phenotype has become an important consideration in drug development and clinical practice. To understand how naturally occurring genetic variation in CYP2D6 causes phenotypic variation, we determined the genotype-phenotype map for CYP2D6 with regard to two single nucleotide polymorphisms (SNPs) of CYP2D6 at the molecular, cellular, and organismal phenotypic levels, and further calculated the intra-genotype individual variability.

2. EXPERIMENTAL SECTION 2.1 Human Liver Samples. As previously described 26, 105 normal human liver samples from Chinese patients were obtained from the First Affiliated Hospital and People's Hospital of Zhengzhou University between March 2012 and July 2014 and informed consent was obtained from each volunteer. Due to the limited size of some samples, genotype, protein content, microsomal protein per gram of liver (MPPGL), and metabolic activity were simultaneously determined for only 90 samples. This research was conducted in accordance with the Declaration of Helsinki. Approvals for tissue collection and in vitro xenobiotic metabolism studies were obtained from the Medical Ethics Committee


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of Zhengzhou University. Liver function was determined by histological examination. Liver tissue samples were stored in liquid nitrogen within 30 min of resection. 2.2 Genotyping of CYP2D6. Genomic DNA was isolated from human liver tissue using a genomic DNA purification kit (Beijing Com Win Biotech Co., Ltd., China). Two SNPs of CYP2D6 were detected. One SNP is 100C>T (rs1065852) genotyped by PCR sequencing. The other is 1661G>C (rs1058164) determined by mass spectrometry performed by the LIUHE HUADA Genomics Technology Co., Ltd, Beijing, China. 2.3 Human liver microsomes (HLMs). HLMs were prepared by differential centrifugation and stored at -800C as previously described

27

. Total HLM protein concentrations were determined by the Bradford

method 28. The MPPGL contents were measured as previously described 27. 2.4 Quantification of CYP2D6 protein by LC–MS/MS. A QconCAT protein consisting of 57 stable isotope-labeled peptides from 21 drug metabolizing enzymes (including CYP2D6) in which two or three peptides were selected for each targeted protein was employed to quantify these proteins in HLMs 29. Human liver microsomal proteins and the recombinant QconCAT protein were digested with trypsin after denaturation, reduced, and alkylated. The peptides ASGNLIPQEK and TILDELVQR were chosen to determine protein content by a nano-high performance liquid chromatography coupled to multiple reaction monitoring MS analysis as described in our previous work

30

. The concentration of CYP2D6

protein was determined by nano-LC-multiple reaction monitoring MS using an easy nano-LC (Thermo Fisher Scientific Inc., Waltham, MA, USA) coupled to a TSQ vantage

TM

triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc.,

Waltham, MA, USA).



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2.5 Molecular phenotype. The molecular phenotype is defined as the clearance of drug per pmol CYP2D6 (CLP). The CLP for every subject was determined from the clearance at microsomal level (CLM) divided by the corresponding individual CYP2D6 protein content. 2.6 Cellular phenotype. The cellular phenotype included sub-cellular (microsomal), tissue, and organ phenotypes. The sub-cellular phenotype was defined as CLM, which was determined by measuring the rate of the following reaction with eight substrate concentrations (0.625–960 µM dextromethorphan). Incubation mixtures contained HLMs (0.2 mg protein ml-1), 100 mM phosphate buffer (pH 7.4) with 1 mM NADPH and different concentrations of dextromethorphan. Optimal incubation time was 20 min. Reactions were terminated by adding 10 µl perchloric acid. Metabolites were identified by HPLC-UV.

The Michaelis–Menten constant (Km) and maximum reaction rate (Vmax)

of CYP2D6 was determined by nonlinear regression analysis using GraphPad Prism 5.04 (GraphPad Inc., La Jolla, CA, USA). CLM was calculated based on the ratio of Vmax-to-Km. The tissue phenotype was defined as the clearance in liver tissue (CLLT). According to the values for MPPGL determined above, the CLLT of each genetically different individual was obtained by multiplying each individual MPPGL by the corresponding individual CLM. The organ phenotype was defined as the clearance in liver (CLL). According to the actual body weight (BW) given for each individual, the liver weight (LW) was calculated by multiplying the liver volume (LV) by the liver density, where LV (ml) = 12.5× BW (kg) + 536.4 31 and the liver density was 1.001 g ml-1


32

. The CLL of each

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individual was determined by multiplying each individual LW/BW by individual CLLT. 2.7 Organismal phenotype. The organismal phenotype was defined as the clearance in vivo (CLH). For the extrapolation of the CLH, the values of cardiac output (CO) were determined from data for normal Han Chinese males (n = 783) and females (n = 805); mean values from each group were selected according to the age and gender of donors used in this study 33

. The values of hepatic blood flow (QH) were determined as 24.5% 34 of the CO. The

plasma unbound fraction (fu,p) and ratio of the drug concentration in blood to plasma (RB) for dextromethorphan was obtained from published studies

35, 36

. According to

the QH, fu,p and RB, the CLH of different genotypes were extrapolated using the Bias-Corrected Conventional in vitro–in vivo extrapolation method 21. 2.8 Statistical Analyses. Statistical analysis was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). The normality of the data distribution was assessed using the method of Kolmogorov-Smirnov and Shapiro-Wilk. Because most data sets were not normally distributed, the Mann-Whitney U test was used for pairwise comparisons, and the Kruskal-Wallis H test was applied for multiple pairwise comparisons. The r was determined using non-parametric Spearman rank correlation analysis. A P-value T



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(rs1065852) located in exon 1 of CYP2D6, which will result in substitution of the amino acid (P34S). The frequency of the wild-type, heterozygous, and homozygous genotype was 31.1%, 22.2%, and 46.7%, respectively. The other SNP is 1661G>C (rs1058164) located in exon 3 and is a synonymous mutation (V136V). The frequency of wild-type, heterozygous, and homozygous genotype was 50.6%, 37.1%, and 12.3%, respectively. 3.2 Molecular phenotype. The two SNPs had a significant influence on the protein content of CYP2D6 (Figure 1A). The content of CYP2D6 for subjects with 100TT (15.11 pmol mg-1) was significantly lower than 100CC (21.65 pmol mg-1) and 100CT (21.68 pmol mg-1) subjects. The content of CYP2D6 was not different between 100CC and 100CT individuals. In contrast to 100C>T, the content of CYP2D6 for 1661GG individuals (14.57 pmol mg-1) was significantly lower than that of 1661GC (21.94 pmol mg-1) and 1661CC (21.65 pmol mg-1) subjects. The protein content of individuals within each group showed marked individual variation. The molecular phenotype is defined as the clearance of drug per pmol CYP2D6 (CLP). The two SNPs of CYP2D6 had a significant influence on the CLP (Figure 1B). The CLP of CYP2D6 for subjects with 100TT (0.15 µl min-1pmol-1) was significantly lower than that of subjects with 100CC (0.22 µl min-1pmol-1) and 100CT (0.24 µl min-1pmol-1), while the CLP of CYP2D6 was not different between 100CC and 100CT individuals. For 1661G>C, only the CLP of CYP2D6 in 1661GC (0.24µl min-1pmol-1) individuals was significantly greater than that of 1661GG (0.12µl min-1pmol-1) individuals. From these results we can draw the conclusion that different genotypes lead to different molecular phenotypes. Meanwhile, it is important to note that the molecular phenotype was also different among the subjects with same genotype.


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3.3 Cellular phenotype. The cellular phenotype encompasses the sub-cellular (microsomal) phenotype, the tissue phenotype, and the organ phenotype. The sub-cellular phenotype is defined as CLM and was influenced by SNPs of CYP2D6 (Figure 2). For 100C>T, the homozygous mutant individuals had the lowest CLM. Statistical analysis showed that the CLM was markedly lower for 100TT individuals (1.81 µl min-1mg-1) than 100CC (5.32 µl min-1mg-1) and 100CT (4.42 µl min-1mg-1) individuals. The 1661G>C also influenced the CLM and the wide-type individuals had the lowest CLM. the CLM for 1661GG individuals (1.59 µl min-1mg-1) was significantly lower than 1661GC (4.79 µl min-1mg-1) and 1661CC (4.91 µl min-1mg-1) individuals. The greater differences among the groups in the CLM may be due to the additive effects caused by CLP and protein content. The microsomal protein per gram of liver (MPPGL) was determined for 90 subjects, and the values for MPPGL levels were significantly different between genetically different individuals (Figure 3A). For CYP2D6 100C>T, the values of MPPGL for wild-type, heterozygous, and homozygous individuals were 46.2 (20.5～127.9), 37.2 (9.9～56.2), and 30.5 (15.2～74.2) mg g-1, respectively. The values of MPPGL for 100CC individuals were observably higher than those found for 100TT individuals. For CYP2D6 1661G>C, the values of MPPGL of wild-type, heterozygous, and homozygous individuals were 30.2 (15.2～127.9), 40.4 (9.9～116.5), and 47.2 (21.3～98.0) mg g-1, respectively. The values of MPPGL for 1661GG individuals were observably lower than those in 1661GC and 1661CC individuals. The tissue phenotype is defined as the clearance in liver tissue (CLLT). According to the values for MPPGL determined above, the CLLT of each genetically different individual was obtained by multiplying each individual MPPGL by the corresponding



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individual CLM. The CLLT values of 100TT individuals (73.14 µl min-1g-1) were dramatically lower than those of 100CC (243.52 µl min-1g-1) and 100CT (197.27 µl min-1g-1) individuals, the CLLT values of 1661GG (65.19 µl min-1g-1) were dramatically lower than 1661GC (207.34 µl min-1g-1) and 1661CC (244.29 µl min-1g-1) individuals (Figure 3B). Furthermore, it is worthwhile to note that the intra-genotype individual variabilities in CLLT for all genotypes were larger than CLM, which may be caused by variability in MPPGL. The body weight (BW) and the liver weight (LW) of genetically different individuals was determined (Figure 4A). The values of BW of 100CC, 100CT, and 100TT individuals were 64.0 (47.0～92.0), 64.5 (45.0～85.0), and 64.0 (30.0～89.0) kg, respectively. The values of LW of 100CC, 100CT, and 100TT individuals were 1337.2 (1125.0～1688.1), 1343.7 (1100.0～1600.5), and 1337.2 (912.3～1650.6) g, respectively. For 1661G>C, the values of BW of wild-type, heterozygous, and homozygous subjects were 64.0 (30.0～89.0), 64.0 (45.0～92.0), and 64.0 (50.0～ 82.5) kg, respectively. The values of LW of wild-type, heterozygous, and homozygous subjects were 1337.2 (912.3 ～ 1650.6), 1337.2 (1100.0 ～ 1688.1), and 1337.7 (1162.6～1569.2) g, respectively. The organ phenotype was defined as clearance in liver (CLL). According to the BW and LW determined above, the CLL of different genotypes was obtained by multiplying each individual LW/BW by individual CLLT. The CLL values of 100TT individuals (1.65 µl min-1kg-1) were dramatically lower than 100CC (5.15 µl min-1kg-1) and 100CT (4.08 µl min-1kg-1) individuals, and the CLLT values of 1661GG (1.36 µl min-1kg-1) were dramatically lower than 1661GC (4.30 µl min-1kg-1) and 1661CC (5.68 µl min-1kg-1) individuals (Figure 4B). Because there was no statistical difference for LW/BW among the different groups, the intra-genotype individual variabilities in


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CLL for all genotypes were similar to the CLLT. 3.4 Organismal phenotype. The values of cardiac output (CO) were determined based on age and gender, and the values of hepatic blood flow (QH) were determined based on the CO. The values of CO of 100CC, 100CT, and 100TT individuals were 5.1 (4.9～6.7), 5.4 (4.9～5.9), and 5.4 (4.9～6.7) L min-1, respectively. The values of QH of 100CC, 100CT, and 100TT individuals were 1259.3 (1205.4～1629.3), 1315.7 (1205.4～1438.2), and 1315.7 (1205.4～1629.3) ml min-1, respectively (Figure 5A). For 1661G>C, the values of CO of wild-type, heterozygous, and homozygous individuals were all 5.1 (4.9～6.7) L min-1. The values of QH of wild-type, heterozygous, and homozygous individuals were all 1259.3 (1205.4～1629.3) ml min-1. The organism phenotype is defined as clearance in vivo (CLH). The plasma unbound fraction (fu,p) and ratio of the drug concentration in blood to plasma (RB) for dextromethorphan was 0.5 and 0.55, respectively

35, 36

. According to the QH

determined above, the fu,p and RB, the CLH of the different genotypes was extrapolated using the Bias-Corrected Conventional in vitro–in vivo extrapolation method (Figure 5B). We found that the CLH values of 100TT individuals (2636.2 ml min-1) were significantly lower than 100CC (8790.4 ml min-1) and 100CT (7254.7 ml min-1) individuals, the CLH values of 1661GG (2414.4 ml min-1) were markedly lower than 1661GC (7913.3 ml min-1) and 1661CC (8915.7 ml min-1) individuals. 3.5 Diversity in phenotypes at different levels. From genotype to phenotype, generally speaking, the CYP2D6 100C>T substitution led to decreased clearance, and the CYP2D6 1661G>C substitution led to increased clearance at each level. Compared with wild type (100CC or 1661GG), the changes in the molecular phenotype (CLP) for individuals with substitutions were not



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obvious (Figure 6). For example, the molecular phenotype of 100CT was basically unchanged, while the 100TT showed an approximate decrease of 30.0%. For 1661G>C, the molecular phenotype of 1661GC increased by about 50.0%, while the 1661CC increased to 1.66-fold. The fold change of organismal phenotype (CLH) for mutant individuals was relatively larger than that for the molecular phenotype. The CLH of 100CT and 100TT were reduced by 18.6% and 70.4%, respectively. The CLH of 1661GC and 1661CC increased by 3.28- and 3.69-fold, respectively. For cellular phenotypes (CLM, CLLT, and CLL), changes were basically the same for 100CT, 100TT, and 1661GC individuals, demonstrating a change of 0.80, 0.30, and 3.15-fold, respectively. The changes in CLM (3.10-fold), CLLT (3.75-fold), and CLL (4.17-fold) for 1661CC were different. In a word, the CYP2D6 phenotypes at different levels were diverse. 3.6 Correlations among phenotypes at different levels. As shown in Table 1, although the phenotypes at different levels are highly correlated with each other (r>0.6, PT and 1661G>C on the protein content (A) and CLP (B) of CYP2D6. Figure 2. Effects of CYP2D6 100C>T and 1661G>C on the CLM of CYP2D6. Figure 3. Effects of CYP2D6 100C>T and 1661G>C on the MPPGL (A) and CLLT of CYP2D6 (B). Figure 4. Effects of CYP2D6 100C>T and 1661G>C on the LW/BW (A) and CLL of CYP2D6 (B). Figure 5. Effects of CYP2D6 100C>T and 1661G>C on the QH (A) and CLH of CYP2D6 (B). Figure 6. The diversity in CYP2D6 phenotypes at different levels (100CC, n=28; 100CT, n=20; 100TT, n=42; 1661GG, n=45; 1661GC, n=33; 1661CC, n=11). Figure 7. The coefficient of variation (CV) of CLP, CLM, CLLT, CLL and CLH of CYP2D6 with different genotypes (100CC, n=28; 100CT, n=20; 100TT, n=42; 1661GG, n=45; 1661GC, n=33; 1661CC, n=11).

Table 1. The Correlation among the phenotypes at different levels CLP a

CLM b

CLLT c

CLL d


CLH e


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(µl min-1pmol-1)

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(µl min-1mg-1)

(µl min-1g-1) (µl min-1kg-1)

(ml min-1)

CLP a 1

0.756***

0.659***

0.647***

0.660***

CLM b

1

0.895***

0.895***

0.892***

1

0.997***

0.994***

1

0.987***

CLLT c CLL d CLH e

1

a

The molecular phenotype or clearance at the protein level.

b

The sub-cellular phenotype or clearance at microsomal level.

c

The tissue phenotype or clearance at liver tissue level.

d

The organ phenotype or clearance at liver level.

e

The organism phenotype or clearance at in vivo level.

CLP was the molecular phenotype; CLM, CLLT, and CLL were the cellular phenotypes; CLH was the organismal phenotype. Data obtained from 90 subjects; *PT (CC, n=28; CT, n=20; TT, n=42); 1661 G>C (GG, n=45; GC, n=33; CC, n=11). The Mann-Whitney U test was used for statistical analysis comparison.

Figure 3.



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CLLT is the tissue phenotype which is one of the cellular phenotypes determined from clearance at microsomal level multiplied by MPPGL. MPPGL, microsomal protein per gram of liver. Black horizontal lines represent medians with the interquartile range. 100 C>T (CC, n=28; CT, n=20; TT, n=42); 1661 G>C (GG, n=45; GC, n=33; CC, n=11). The Mann-Whitney U test was used for statistical analysis comparison.

Figure 4.

CLL is the organ phenotype which is one of the cellular phenotypes determined from clearance at liver tissue level multiplied by LW/BW. LW: liver weight, BW: body weight. Black horizontal lines represent medians with the interquartile range. 100 C>T (CC, n=28; CT, n=20; TT, n=42); 1661 G>C (GG,

n=45;

GC,

n=33;

CC,

n=11).

The

Mann-Whitney

statistical analysis comparison.


U

test

was

used

for

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Figure 5.

⁄

CLH is the organismal phenotype calculated using the well-stirred model: ,⁄ .

,

CC: Correction coefficient; QH: hepatic blood flow; CLL: clearance at liver level; fu,p: plasma unbound fraction; RB: ratio of the drug concentration in blood to plasma. Black horizontal lines represent medians with the interquartile range. 100 C>T (CC, n=28; CT, n=20; TT, n=42); 1661 G>C (GG, n=45; GC, n=33; CC, n=11). The Mann-Whitney U test was used for statistical analysis comparison.

Figure 6.

CLP: molecular phenotype or clearance at the protein level; CLM: sub-cellular phenotype or clearance at microsomal level; CLLT: tissue phenotype or clearance at liver tissue level; CLL: organ phenotype or clearance at liver level; CLH: organism phenotype or clearance at in vivo level. CLP was the molecular phenotype; CLM, CLLT, and CLL were the cellular phenotypes; CLH was the organismal phenotype. Fold change was the ratio of the median of the mutant and wild-type. “*’’, “**’’, and “***” indicate significant differences from 100CC or 1661GG (P

From Genotype to Phenotype - American Chemical Society

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