Downregulation of sulfotransferase expression and activity in diseased human livers

Abstract

Sulfotransferase (SULT) function has been well studied in healthy human subjects by quantifying mRNA and protein expression and determining enzyme activity with probe substrates. However, it is not well known if sulfotransferase activity changes in metabolic and liver disease, such as diabetes, steatosis, or cirrhosis. Sulfotransferases have significant roles in the regulation of hormones and excretion of xenobiotics. In the present study of normal subjects with nonfatty livers and patients with steatosis, diabetic cirrhosis, and alcoholic cirrhosis, we sought to determine SULT1A1, SULT2A1, SULT1E1, and SULT1A3 activity and mRNA and protein expression in human liver tissue. In general, sulfotransferase activity decreased significantly with severity of liver disease from steatosis to cirrhosis. Specifically, SULT1A1 and SULT1A3 activities were lower in disease states relative to nonfatty tissues. Alcoholic cirrhotic tissues further contained lower SULT1A1 and 1A3 activities than those affected by either of the two other disease states. SULT2A1, on the other hand, was only reduced in alcoholic cirrhotic tissues. SULT1E1 was reduced both in diabetic cirrhosis and in alcoholic cirrhosis tissues, relative to nonfatty liver tissues. In conclusion, the reduced levels of sulfotransferase expression and activity in diseased versus nondiseased liver tissue may alter the metabolism and disposition of xenobiotics and affect homeostasis of endobiotic sulfotransferase substrates.

Figures

Fig. 1.

Expression and activity of SULT1A1 in nonfatty (control) and diseased human livers. Each data point represents a single tissue (average of determinations) categorized by gender and disease type (n = 20 for nonfatty; n = 13–14 for steatosis; n = 4 for diabetes; n = 21–22 diabetic cirrhosis; and n = 17–22 for alcohol cirrhosis). Tissue from female patients is displayed in red; from male patients, in blue. *Statistically significant differences (P < 0.05) between nonfatty and diseased livers. #Statistically significant differences (P < 0.05) between alcohol cirrhosis and other groups. (A) Enzyme activity was determined by incubating 4 μM pNP with human liver cytosols for 30 minutes in the presence of the 35S-labeled cofactor PAPS. (B) Protein expression of SULT1A1 was quantified by Western blotting analysis in human livers. (C) Messenger RNA expression of SULT1A1 was quantified by branched DNA signal amplification assay (Affymetrix Inc., Santa Clara, CA). (D) Membranes loaded with 40 μg protein and probed with anti-SULT1A1 antibody. Lane numbers refer to sample ID as noted in Supplemental Table 1.

Fig. 2.

Expression and activity of SULT2A1 in nonfatty (control) and diseased human livers. Each data point represents a single tissue (average of determinations) categorized by gender and disease type (n = 20 for nonfatty; n = 13–14 for steatosis; n = 4 for diabetes; n = 21–22 diabetic cirrhosis; and n = 17–22 for alcohol cirrhosis). Tissue from female patients is displayed in red; from male patients, in blue. #Statistically significant differences (P < 0.05) between alcohol cirrhosis and other groups. (A) Enzyme activity was determined by incubating 10 μM DHEA with human liver cytosols for 30 minutes in the presence of the 35S-labeled cofactor PAPS. (B) Protein expression of SULT2A1 was quantified by Western blotting analysis in human livers. (C) Messenger RNA expression of SULT2A1 was quantified by branched DNA signal amplification assay (Affymetrix Inc.). (D) Membranes loaded with 40 μg protein and probed with anti-SULT2A1 antibody. Lane numbers refer to sample ID as noted in Supplemental Table 1.

Fig. 3.

Expression and activity of SULT1E1 in nonfatty (control) and diseased human livers. Each data point represents a single tissue (average of determinations) categorized by gender and disease type (n = 20 for nonfatty; n = 13–14 for steatosis; n = 4 for diabetes; n = 21–22 diabetic cirrhosis; and n = 17–22 for alcohol cirrhosis). Tissue from female patients is displayed in red; from male patients, in blue. *Statistically significant differences (P < 0.05) between nonfatty and diseased liver groups. +Statistically significant differences (P < 0.05) between steatosis and other groups. (A) Enzyme activity was determined by incubating 20 nM 3H-labeled estradiol with human liver cytosols for 30 minutes in the presence of the 35S-labeled cofactor PAPS. (B) Protein expression of SULT1E1 was quantified by Western blotting analysis in human livers. (C) Messenger RNA expression of SULT1E1 was quantified by branched DNA signal amplification assay (Affymetrix Inc.). (D) Membranes loaded with 40 μg protein and probed with anti-SULT1E1 antibody. Lane numbers refer to sample ID as noted in Supplemental Table 1.

Fig. 4.

Expression and activity of SULT1A3 in nonfatty (control) and diseased human livers. Each data point represents a single tissue (average of determinations) categorized by gender and disease type (n = 20 for nonfatty; n = 13–14 for steatosis; n = 4 for diabetes; n = 21–22 diabetic cirrhosis; and n = 17–22 for alcohol cirrhosis). Tissue from female patients is displayed in red; from male patients, in blue. *Statistically significant differences (P < 0.05) between nonfatty and diseased livers. +Statistically significant differences (P < 0.05) between steatosis and other groups. #Statistically significant differences (P < 0.05) between alcoholic cirrhosis and other groups. (A) Enzyme activity was determined by incubating 10 μM dopamine with human liver cytosols for 30 minutes in the presence of the 35S-labeled cofactor PAPS. (B) Protein expression of SULT1A3 was quantified by Western blotting analysis in human livers. (C) Messenger RNA expression of SULT1A3 was quantified by quantitative real-time polymerase chain reaction using a Roche LightCycler 480 System (Roche Applied Science, Mannheim, Germany). (D) Membranes loaded with 40 μg protein and probed with anti-SULT1A3 antibody. Lane numbers refer to sample ID as noted in Supplemental Table 1.

Fig. 5.

Total and relative sulfonation of SULT1A1, SULT2A1, SULT1E1, and SULT1A3 in non-fatty (control) and diseased human livers. Total sulfotransferase activity is indicated by colored bars in each tissue type. The percentage of sulfotransferase activities of four major isoforms were calculated for nonfatty (control) group.

Fig. 6.

PAPSs2 mRNA expression in humans in nonfatty (control) and diseased human livers. PAPSs2 mRNA expression was quantified by branched DNA signal amplification assay (Affymetrix Inc.). *Statistically significant differences (P < 0.05) between nonfatty and diseased livers.