Significantly decreased and more variable expression of major CYPs and UGTs in liver microsomes prepared from HBV-positive human hepatocellular carcinoma and matched pericarcinomatous tissues determined using an isotope label-free UPLC-MS/MS method

Abstract

Purpose: To determine the liver expression of cytochrome P450 (CYPs) and uridine 5'-diphosphate-glucuronosyltransferases (UGTs), the major phase I and II metabolism enzymes responsible for clearance and detoxification of drugs, xenobiotic and endogenous substances.

Methods: A validated isotope label-free method was established for absolute and simultaneous quantification of 9 CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D, 2E1 and 3A4) and 5 UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes using LC-MS/MS.

Results: The LC-MS/MS method displayed excellent dynamic range (at least 250-fold) and high sensitivity for each of the signature peptides with acceptable recovery, accuracy and precision. The protein expression profile of CYP and UGT isoforms were then determined in match microsomes samples prepared from patients with HBV-positive human hepatocellular carcinoma (HCC). In the tumor microsomes, the average absolute amounts of 8 major CYP isoforms (except CYP2C19) and 3 UGT isoforms (UGT1A1, UGT1A4 and UGT2B7) were decreased significantly (p < 0.05), whereas UGT1A6 and UGT1A9 levels were unchanged (p > 0.05). In addition, among isoforms with altered expression, 6 of 8 CYP isoforms and all three UGT isoforms were much more variable in tumor microsomes. Lastly, the importance of CYP3A4 was greatly diminished whereas the importance of UGT1A6 was enhanced in tumor microsomes.

Conclusion: The use of an isotope label-free absolute quantification method for the simultaneous determination of 9 CYPs and 5 UGTs in human liver microsomes reveals that expression levels of CYPs and UGTs in human liver are severely impact by HCC, which could impact drug metabolism, disposition and pharmacotherapy.

Fig. 1

Overlaps of three MRM chromatograms of a digested individual human liver microsomes, standard calibrant and the digested HLMs spiked with the calibrant.

Fig. 2

(A-I) Protein expression level of nine CYP isoforms in human liver microsomes prepared from tumor tissues (tHLMs-individual) and matched pericarcinomatous tissues (nHLMs-individual) of 15 patients with HCC. Each data point represents the average of two determinations using two MRM transitions, and data are presented as mean±SD. Paired-samples T-test was used for data analysis. “*” denotes statistical significance (p<0.05).

Fig. 3

Average expression levels of nine CYPs in 15 tumor tissues and pericarcinomatous tissues. The error bar represents the mean±SD calculated from the protein amount of each isoform in 15 donors. Mann-Whitney U test was used for data analysis. “*” denotes statistical significance (p<0.05).

Fig. 4

(A-E) Protein expression level of five UGT isoforms in human liver microsomes prepared from tumor tissues (tHLMs-individual) and matched pericarcinomatous tissues (nHLMs-individual) of 15 patients with HCC. Each data point represents the average of two determinations using two MRM transitions, and data are presented as mean±SD. Paired-samples T-test was used for data analysis. “*” denotes statistical significance (p<0.05).

Fig. 5

Average expression level of five UGTs in 15 tumor tissues and pericarcinomatous tissues. The error bar represents the standard deviation calculated from the protein amount of each isoform in 15 donors. Mann-Whitney U test was used for data analysis. “*” denotes statistical significance (p<0.05).

Fig. 6

Protein expression levels of nine CYPs (A) and five UGTs (B) in rHLM-pooled, nHLMs-pooled and tHLMs-pooled. Each data point represents the average of two determinations using two MRM transitions, and data are presented as mean±SD. Paired-samples T-test was used for data analysis. “*” denotes statistical significance (p<0.05).

Fig. 7

(A) A comparison of CYPs and UGTs expression levels in rHLMs-pooled observed in the present study (Observed Values) with values reported previously (Reported Values). (B) The correlation plots of the observed and reported values of nine CYP isoforms. (C) The correlation plots of the observed and reported values of four UGT isoforms.

Fig. 8

Digestion time profiles for signature peptides of CYP isoforms in recombinant CYP enzymes. Values shown represent percentages of the maximum concentration for each peptide.