Tissue Distribution Of The Human Sult1a Subfamily

A number of studies have employed metabolic probes and immunohistochemistry techniques to show that SULT1A SULTs exhibit a wide tissue distribution, including liver, lung, brain, skin, platelets, gastrointestinal tissues, and kidney (Cappiello et al., 1990; Heroux et al., 1989; Hume and Coughtrie, 1994; Kudlacek et al., 1995; Zou et al., 1990). However, it was not until 1995, when SULT1A2 was first cloned (Ozawa et al., 1995), that we definitively knew the SULT1A subfamily contained three members, which share >90% sequence identity at the amino acid level. This high sequence identity together with the overlapping substrate specificity of SULT enzymes has compromised the use of immunohistochemistry and metabolic probes to study their tissue and cellular localization. While the early studies on SULT 1A localization relied heavily on the predictive nature of p-nitrophenol and dopamine-specific SULT activity assays in tissues, they nonetheless gave us significant insight into the tissue-specific expression patterns of these enzymes (Weinshilboum, 1986). For example, it was shown that blood platelets exhibited high SULT1A1 activity (TS-PST; phenol SULT activity) and that it could be correlated with the sulfonation profile observed in other tissues such as brain, liver, kidney, and small intestine (Abenhaim et al., 1981; Anderson et al., 1981; Glatt et al., 2001; Hart et al., 1979; Sundaram et al., 1989; Young et al., 1985). In contrast, no correlation was found between SULT1A3 activity (TL-PST; monoamine SULT activity) in platelets and that observed in brain, liver, or small intestine tissue samples (Campbell et al., 1987; Sundaram et al., 1989; Young et al., 1985). Such studies were also the first to demonstrate the large interindividual differences in phenol SULT activity within the human population and the importance of genetic polymorphisms in controlling SULT1A activity in human tissues (Price et al., 1988, 1989; Weinshilboum, 1992).

It has been possible to show, using an array of approaches such as hybridization histochemistry, immunoblotting (Figure 10.2), and RT-PCR analysis, that SULT1A1, SULT1A2, and SULT1A3 vary in their tissue localization. For example, in the adult liver, SULT1A1 is expressed at high levels, but SULT1A2 and SULT1A3 are almost undetectable (Eisenhofer et al., 1999; Heroux et al., 1989; Richard et al., 2001; Windmill et al., 1997, 1998). In our laboratory, we have used hybridization histochemistry, immunohistochemistry, and immunoblot analysis to study the localization of SULT1A1 and SULT 1 A3. For localization studies using hybridization histochemistry, we have employed a full-length (1155 bp) SULT1A1 cDNA and a 198 bp PCR fragment (1175-1372) specific for the 3' end of the untranslated region of SULT1A3. Hybridization studies showed that the SULT1A3 probe was specific, but the full-length SULT1A1 probe detected all SULT1A subfamily members (Windmill et al., 1998). For the identification of SULT1A-specific protein, a polyclonal antibody was raized in the rabbit against the E. coft-expressed recombinant human SULT1A1 protein (amino acids 31-286). This antibody was not specific for the SULT1A1 enzyme but was able to immuno-react with all three proteins of the SULT1A subfamily. However, on polyacrylamide gel electrophoresis and subsequent immunoblotting, it is possible to distinguish between the three isozymes based on differential migration on the gel. The presence of both SULT1A mRNA and protein was observed in liver, gastrointestinal tract (stomach, small intestine, and colon), and lung. From immunoblotting analysis, SULT1A1 was predominant in the liver, and high levels of mRNA and protein were observed across the acinus. SULT1A1 and SULT1A3 protein could be detected on immunoblots in cytosolic fractions of stomach, small intestine, and colon (Windmill et al., 1998). From the histological studies, SULT1A mRNA and protein were detected in epithelial cells lining the lumen of the stomach and the gastric pits and in the epithelial cells lining the lumen surface and the crypts of Lieberkuhn of the small intestine and colon. Similarly, SULT1A1 and SULT1A3 were detected in lung cytosols, and histological studies using hybridization histochemistry and immunohistochemistry localized SULT1A mRNA and protein to the epithelial cells of the respiratory bronchioles. The widespread localization of SULT1A mRNA and protein throughout the human gastrointestinal tract and lung suggests they may play a significant role in the extrahepatic detoxification and activation of drugs and xenobiotics (Windmill et al., 1998; Windmill, K.F., Hall, P.M., McManus, M.E., unpublished data).

Enzyme kinetic data obtained from purified recombinant SULT protein have enabled the modeling of catalytic function in tissue cytosols by choosing substrate concentrations that are close to Km values of the individual SULT1A isozymes. For example, Richard et al. (2001) used 3.3 ^M p-nitrophenol and 4.7 ^M dopamine, values close to the Km values of SULT1A1 and SULT1A3 for the respective substrates, to assess the sulfonation of these compounds in human fetal and adult tissues. The authors showed a correlation between the sulfonation of 3,3'-T2 and that of p-nitrophenol, but not to dopamine sulfonation, and concluded that the enzyme responsible for 3,3'-T2 in the tissues tested was SULT1A1 (Richard et al., 2001). Although immunohistochemical detection of tissue sections was not specific for either isoform, immunoblotting of tissue cytosols was able to distinguish the enzymes based on their differential migration on SDS-PAGE. Together with kinetic data, it was revealed that in the fetal brain, sulfonation of dopamine and SULT1A3 protein levels were very low in all areas tested. The sulfonation of 0.1 ^M 3,3'-T2 in the brain was shown to be highest in the choroid plexus of the lateral ventricle, and the investigators suggested that the high expression of SULT1A1 in this region could be a mechanism of defense against portally transported toxins, as this region is the most highly vascularized in the developing brain (Richard et al., 2001). Earlier studies also showed SULT1A activity in the adult and fetal brain (Richard et al., 2001; Whitte-more et al., 1985, 1986; Young et al., 1985). Young et al. (1985) reported highest dopamine and phenol SULT activity in the cerebral cortex. Phenol SULT activity was also apparent in the anterior pituitary, and activity was 6.5 times higher than in the parts of the brain that are of neuronal origin.

One interesting observation made by Richard et al. (2001) was the developmental expression difference apparent between SULT1A1 and SULT1A3 in lung and liver tissues. Assessing the dopamine and thyroid hormone sulfonation ability of fetal tissues, it was found that high liver SULT1A1 activity was generally retained into adulthood; however, lung activity reduced approximately 10-fold toward 3,3'-T2 (Gilissen et al., 1994; Richard et al., 2001). Sulfonation activity of dopamine was high in the fetal liver and lung but reduced significantly in the postnatal tissues (Pacifici et al., 1993; Richard et al., 2001). Protein levels confirmed these results, suggesting that SULT1A1 and 1A3 are abundantly expressed in the fetal liver and that the SULT 1 A3 enzyme almost disappears in the adult tissue (Richard et al., 2001). This same pattern has also been observed for SULT1A3 in the kidney (Cap-piello et al., 1991). These data suggest an important role for these two SULTs in the protection of the fetus from exogenous toxins and in the homeostasis of hormones such as dopamine and iodothyronines. Immunoblotting of placenta cytosols revealed positive staining for both SULT1A1 and SULT1A3 enzymes, mainly from the cotyledon region where they may have an important role in the xenobiotic metabolism of potentially harmful chemicals entering the fetal circulation from the maternal side (Heroux et al., 1989; Stanley et al., 2001). No immunoreactive band was observed for SULT1A2 in this tissue. Stanley et al. (2001) also showed activities for both dopamine and p-nitrophenol in the placenta, and again p-nitrophenol sul-fonation was correlated to that of 3,3'-T2. The available data suggest that SULTs may play a significant role in the phase II metabolism in the placenta, as other conjugating enzymes such as UDP-glucuronosyltransferases seem to be expressed at minimal levels in this tissue.

Another reproductive tissue that shows SULT1A1 activity is the endometrium, where levels do not seem to change according to the menstrual cycle; this is different from the pattern observed with the estrogen SULT (SULT1E1), whose expression appears to be regulated by progesterone (Falany and Falany, 1996). Although SULT1A1 and SULT1A3 have been found only at low levels in mammary gland tissue in immunohistochemical studies, it has been shown with activity assays and immunoreactivity studies that most breast cancer cell lines, including estrogen receptor positive and negative lines, express high levels of both SULT enzymes (Falany and Falany, 1996; Windmill et al., 1998). SULT1A RT-PCR and activity studies have also confirmed the presence of SULT1A1 and 1A3 in carcinoma cell lines, including Caco-2, HepG2, keratinocarcinomas, melanomas, fibrosarcomas, osteosarcoma, and osteoblast cells (Baranczyk-Kuzma et al., 1991; Dooley et al., 2000; Dubin et al., 2001; Satoh et al., 2000). It seems that SULT1A3 is the most abundant form found in the human colon carcinoma cell line Caco2, reflecting its dominance in the normal gastrointestinal tract, whereas SULT1A1 represents the most abundantly expressed SULT in human MCF-7 breast carcinoma cells (Falany et al., 1993; Satoh et al., 2000). RT-PCR studies show that SULT1A1 and SULT1A3 are transcribed ubiquitously throughout many epithelial tissues and cell lines (Dooley et al., 2000). One role of the SULT1A isoforms in the skin is the activation of the topically applied hair growth stimulant minoxidil by sulfo-conjugation, and their distribution in epithelial tissues may serve as both a defense and bioactivation mechanism for xeno-biotics entering the body via this route (Dooley et al., 2000). Dooley and colleagues have pointed out that the pattern of SULT1A mRNA expression does not always translate to the formation of protein and have suggested that posttranscriptional and posttranslational modification events may take place. As mentioned above, little is known of the tissue distribution of SULT1A2; however, lower mRNA levels than the other SULT1As are found in liver, kidney, brain, lung, ovary, and some sections of the gastrointestinal tract, and recently SULT1A2 levels have been observed in some bladder tumors (Dooley et al., 2000; Glatt et al., 2001).

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