A high frequency missense SULT1B1 allelic variant (L145V) selectively expressed in African descendants exhibits altered kinetic properties.

1. Human cytosolic sulfotransferase 1B1 (SULT1B1) sulfates small phenolic compounds and bioactivates polycyclic aromatic hydrocarbons. To date, no SULT1B1 allelic variants have been well-characterized. 2. While cloning SULT1B1 from human endometrial specimens, an allelic variant resulting in valine instead of leucine at the 145th amino acid position (L145V) was detected. NCBI reported this alteration as the highest frequency SULT1B1 allelic variant. 3. L145V frequency comprised 9% of 37 mixed-population human patients and was specific to African Americans with an allelic frequency of 25%. Structurally, replacement of leucine with valine potentially destabilizes a conserved helix (α8) that forms the "floor" of both the substrate and PAPS binding domains. This destabilization results in altered kinetic properties including a four-fold decrease in affinity for PAP (3', 5'-diphosphoadenosine). Kms for 3'-phosphoadenosine- 5'-phosphosulfate (PAPS) are similar; however, maximal turnover rate of the variant isoform (0.86 pmol/(min*µg)) is slower than wild-type (WT) SULT1B1 (1.26 pmol/(min*µg)). The L145V variant also displays altered kinetics toward small phenolic substrates, including a diminished p-nitrophenol Km and increased susceptibility to 1-naphthol substrate inhibition. 4. No significant correlation between genotype and prostate or colorectal cancer was observed in patients; however, the variant isoform could underlie specific pathologies in sub-Saharan African carriers.

Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with ß-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity.

Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression.

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERalpha and ERbeta) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenases (17betaHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERalpha-positive luminal MCF7 breast cancer cells. Low levels of ERalpha and ERbeta mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17betaHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17betaHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17betaHSD1, and 17betaHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERalpha, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERbeta, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

Regulation of hepatic sulfotransferase (SULT) 1E1 expression and effects on estrogenic activity in cystic fibrosis (CF).

Cystic fibrosis (CF) is a major genetic disease in Caucasians affecting 1 in 2500 newborns. Hepatobiliary pathology is a major cause of morbidity and mortality in CF second only to pulmonary disease. SULT1E1 activity is significantly elevated, generally 20-30-fold, in hepatocytes of mouse models of CF. SULT1E1 is responsible for the inactivation of beta-estradiol (E2) at physiological concentrations via conjugation with sulfonate. The increase in SULT1E1 activity results in the alteration of E2-regulated protein expression in CF mouse liver. To investigate the mechanism by which the absence of CFTR in human cholangiocytes induces SULT1E1 expression in hepatocytes, a membrane-separated human MMNK-1 cholangiocyte and human HepG2 hepatocyte co-culture system was developed. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in bile duct cholangiocytes but not hepatocytes, whereas SULT1E1 is expressed in hepatocytes but not cholangiocytes. CFTR expression in MMNK-1 cells was inhibited with siRNA by >90% as determined by immunoblot and immunohistochemical analysis. Control and CFTR-siRNA-MMNK-1 cells were co-cultured with HepG2 cells in a Transwell membrane-separated system. After 8h of co-culture, HepG2 cells were removed from exposure to MMNK-1 cells and placed in fresh medium. After 24-48h, expression of SULT1E1 and selected E2-regulated proteins was analyzed in the HepG2 cells. Results demonstrated that SULT1E1 message and activity were selectively induced in HepG2 cells co-cultured with CFTR-deficient MMNK-1 cells. The expression of E2-regulated proteins (IGF-1, GST-P1 and carbonic anhydrase II) was also altered in response to decreased E2 levels. Thus, the loss of CFTR activity in cholangiocytes stimulates the expression of SULT1E1 in hepatocytes by a paracrine mechanism. SULT1E1 expression in HepG2 cells is inducible by sterol mediated liver-X-receptor (LXR) activation although not by progestins that induce SULT1E1 in the endometrium. SULT1E1 induction in the human cholangiocyte/hepatocyte co-culture system is consistent with and supports the results observed in CF mice. The changes in hepatocyte gene expression affect liver biochemistry and may facilitate the development of CF liver disease.

Increased SULT1E1 activity in HepG2 hepatocytes decreases growth hormone stimulation of STAT5b phosphorylation.

Mouse models of cystic fibrosis (CF) display increased sulfotransferase 1E1 (SULT1E1) activity in hepatocytes of cystic fibrosis transmembrane receptor (CFTR)-deficient animals. SULT1E1 is responsible for the sulfation and inactivation of beta-estradiol (E2) at physiological concentrations. IGF-1 message levels in CFTR(-/-) mouse livers were positively correlated with body weight and negatively correlated with SULT1E1 activity. Growth hormone (GH) is important in the regulation of hepatic IGF-1 expression indicating that E2 levels are involved with GH signaling in hepatocytes. To investigate the effects of E2 and SULT1E1 activity on GH signal transduction in human hepatocytes, SULT1E1 was stably expressed in HepG2 cells. Effects of increased E2 sulfation on the GH signaling pathway and E2-regulated gene expression were examined. Pretreatment of HepG2 cells with 10nM E2 prior to GH stimulation increased STAT5b phosphorylation and IGF-1 expression. In SULT1E1-transfected HepG2 cells, GH-stimulated STAT5b phosphorylation was significantly decreased. E2 treatment had no effect on STAT5b phosphorylation in the absence of GH stimulation. E2 also had no effect on Jak-2 phosphorylation. E2 has an apparent rapid action on increasing GH-stimulated STAT5b phosphorylation that was not attenuated by the estrogen receptor antagonist, ICI 182,780. Physiological levels of E2 in HepG2 cells increase GH stimulation of IGF-1 production apparently through increased phosphorylated STAT5b levels and transcriptional activation of the IGF-1 gene. The enhanced SULT1E1 activity may have a role in inhibiting GH-stimulated STAT5b phosphorylation and IGF-1 synthesis via the sulfation and inactivation of E2.

Repression of CFTR activity in human MMNK-1 cholangiocytes induces sulfotransferase 1E1 expression in co-cultured HepG2 hepatocytes.

Mouse models of cystic fibrosis (CF) indicate that sulfotransferase (SULT) 1E1 is significantly induced in livers of many mice lacking cystic fibrosis transmembrane receptor (CFTR) activity. Increased SULT1E1 activity results in the alteration of estrogen-regulated protein expression in the livers of these mice. In this study, human MMNK-1 cholangiocytes with repressed CFTR function were used to induce SULT1E1 expression in human HepG2 hepatocytes to investigate whether SULT1E1 can be increased in human CF liver. CFTR expression was inhibited in MMNK-1 cholangiocytes using CFTR-siRNA, then the MMNK-1 and HepG2 cells were co-cultured in a membrane-separated Transwell system. Expression of SULT1E1 and selected estrogen-regulated proteins were then assayed in the HepG2 cells. Results demonstrate that inhibition of CFTR expression in MMNK-1 cells results in the induction of SULT1E1 message and activity in HepG2 cells in the Transwell system. The expression of estrogen-regulated proteins including insulin-like growth factor (IGF)-1, glutathione-S-transferase (GST) P1 and carbonic anhydrase (CA) II expression are repressed in the HepG2 cells cultured with the CFTR-siRNA-MMNK-1 cells apparently in response to the increased sulfation of beta-estradiol. Thus, we have shown that co-culture of HepG2 hepatocytes with MMNK-1 cholangiocytes with siRNA repressed CFTR expression results in the selective induction of SULT1E1 in the HepG2 cells. Loss of CFTR function in cholangiocytes may have a paracrine regulatory effect on hepatocytes via the induction of SULT1E1 and the increased sulfation of beta-estradiol. Experiments are presently underway in our laboratory to elucidate the identity of these paracrine regulatory factors.

Positive and negative regulation of human hepatic hydroxysteroid sulfotransferase (SULT2A1) gene transcription by rifampicin: roles of hepatocyte nuclear factor 4alpha and pregnane X receptor.

The effects of rifampicin treatment on SULT2A1 mRNA expression were evaluated in 23 preparations of primary cultured human hepatocytes. In contrast to the consistently occurring induction of CYP3A4, a prototypical pregnane X receptor (PXR) target gene, rifampicin treatment increased SULT2A1 mRNA levels in 12 of the hepatocyte preparations, but it produced little change or even suppression in the others. Transient transfection of HepG2 cells with a series of reporter constructs implicated two SULT2A1 5'-flanking regions as containing rifampicin-responsive information. Each of these regions contained a hepatocyte nuclear factor 4 (HNF4) binding site (at nucleotide [nt] -6160 and -54), as demonstrated by in vitro binding and site-directed mutagenesis. HNF4alpha bound to the HNF4-54 region of the endogenous SULT2A1 gene, as indicated by chromatin immunoprecipitation. Cotransfection of HepG2 cells with pregnane X receptor (PXR) dose-dependently suppressed reporter expression from SULT2A1 constructs containing the HNF4 sites, and rifampicin treatment augmented the suppression. Rifampicin treatment concentration-dependently suppressed SULT2A1 reporter expression at the same concentrations that progressively induced expression from a PXR-responsive CYP3A4 reporter, whereas higher rifampicin concentrations reversed the SULT2A1 suppression. The suppressive effect of rifampicin was diminished, whereas the activating effect was augmented, in HepG2 cells with RNA interference-mediated PXR knockdown. These results suggest that HNF4alpha plays a central role in the control of SULT2A1 transcription and that rifampicin-liganded PXR suppresses SULT2A1 expression by interfering with HNF4alpha activity. By contrast, the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations seems to be mediated through a PXR-independent mechanism.

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene.

Sulfation is important in the metabolism and inactivation of steroidal compounds and hormone replacement therapeutic (HRT) agents in human tissues. Although generally inactive, many steroid sulfates are hydrolyzed to their active forms by sulfatase activity. Therefore, the specific sulfotransferase (SULT) isoforms and the levels of steroid sulfatase (STS) activity in tissues are important in regulating the activity of steroidal and HRT compounds. Tibolone (Tib) is metabolized to three active metabolites and all four compounds are readily sulfated. Tib and the Delta4-isomer are sulfated at the 17beta-OH group by SULT2A1 and the 17-sulfates are resistant to hydrolysis by human placental STS. 3alpha-OH and 3beta-OH Tib can form both 3- and 17-monosulfates as well as disulfates. Only the 3beta-sulfates are susceptible to STS hydrolysis. Raloxifene monosulfation was catalyzed by at least seven SULT isoforms and SULT1E1 also synthesizes raloxifene disulfate. SULT1E1 forms both monosulfates in a ratio of approximately 8:1 with the more abundant monosulfate migrating on HPLC identical to the SULT2A1 synthesized monosulfate. The raloxifene monosulfate formed by both SULT isoforms is sensitive to STS hydrolysis whereas the low abundance monosulfate formed by SULT1E1 is resistant. The benzothiophene sulfates of raloxifene and arzoxifene were hydrolyzed by STS whereas the raloxifene 4'-phenolic sulfate was resistant. These results indicate that tissue specific expression of SULT isoforms and STS could be important in the inactivation and regeneration of the active forms of HRT agents.

Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver.

Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65-75% of total liver BAT activity was found in the cytosol, 15-17% was found in the peroxisomes, and 5-10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT.

Regulation of SULT1E1 expression in Ishikawa adenocarcinoma cells by tibolone.

Tibolone is used therapeutically as a hormone replacement agent and has beneficial effects on osteoporosis and hot flushes as well as libido in post-menopausal women without stimulatory effects in the breast and endometrium. The lack of effect in the endometrium is due in part to the tissue specific sulfation of tibolone and its active metabolites in endometrial tissues. Tibolone is metabolized into 3alpha-OH and 3beta-OH tibolone as well as the Delta4-isomer. Tibolone and the Delta4-isomer bind and activate progesterone and androgen receptors whereas 3alpha-OH and 3beta-OH tibolone activate the estrogen receptors. Human endometrium and Ishikawa endometrial adenocarcinoma cells express SULT1E1 that efficiently sulfates both 3-OH tibolone metabolites and has trace activity with tibolone but no activity with the Delta4-isomer. Treatment of Ishikawa cells with all four tibolone compounds resulted in the induction of SULT1E1 activity similar to the induction by progesterone. The induction of SULT1E1 was inhibited by RU486 indicating a role for the progesterone receptor. Sulfation of the tibolone compounds by Ishikawa cells and Ishikawa cells expressing physiological levels of SULT1E1 activity resulted in the sulfation of tibolone and the 3-OH metabolites but not Delta4-tibolone. These results indicate that the lack of endometrial stimulation involves induction of SULT1E1 and the selective sulfation and inactivation of the estrogenic 3-OH tibolones and interconversion of the tibolone metabolites to generate the progestagenic non-sulfated Delta4-isomer.

Sulfation of raloxifene and 4-hydroxytamoxifen by human cytosolic sulfotransferases.

Raloxifene and 4-hydroxytamoxifen (4-OHT) are important estrogen-related drugs used in the treatment of osteoporosis and breast cancer. Sulfation is involved in the metabolism and inactivation of both compounds in human tissues, although the sulfotransferase (SULT) isoforms involved in their conjugation have not been well described. The ability of seven expressed SULT isoforms to sulfate raloxifene and 4-OHT was investigated. Raloxifene was conjugated by all seven SULT isoforms tested, whereas 4-OHT was conjugated only by SULTs 1A1, 1E1, and 2A1. Characterization of raloxifene and 4-OHT sulfation demonstrates that sulfation can occur at therapeutic concentrations. SULT1E1 displayed the lowest Km (0.2 microM) for 4-OHT sulfation and SULT2A1 the lowest (0.3 microM) for raloxifene sulfation. SULT1E1 was the only isoform exhibiting detectable levels of raloxifene disulfation activity. Modeling of the interactions of raloxifene in the active site of SULT1E1 indicates that both hydroxyl groups of raloxifene can be readily positioned in proximity to the sulfonyl group of 3'-phosphoadenosine 5'-phosphosulfate and the catalytically important His107 residue. Both raloxifene and 4-OHT sulfation activities were detectable in all human liver cytosols tested. 4-OHT sulfation was detected in cytosol prepared from endometrial biopsies of normal women obtained during the proliferative and secretory phases of the same menstrual cycle. In contrast, raloxifene sulfation was detectable only in secretory phase cytosols in association with SULT1E1 activity. In summary, several human SULT isoforms are capable of sulfating raloxifene and 4-OHT. Tissue-specific expression of the individual SULT isoforms may have important roles in the regulation of the activity of these compounds.

Developmental expression of aryl, estrogen, and hydroxysteroid sulfotransferases in pre- and postnatal human liver.

Aryl- (SULT1A1), estrogen- (SULT1E1), and hydroxysteroid- (SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre- and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre- and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre- and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.

Sulfation of tibolone and tibolone metabolites by expressed human cytosolic sulfotransferases.

Tibolone is an important therapeutic agent used in the treatment of menopausal symptoms in many countries and has beneficial effects on menopausal and postmenopausal vasomotor, bone, vaginal and mood symptoms without affecting the endometrial, breast or cardiovascular systems. The rapid metabolism of tibolone to active metabolites including 3alpha-OH-tibolone, 3beta-OH-tibolone and Delta(4)-tibolone may be important in its tissue-specific effects. Sulfation also has a major role in the metabolism and regulation of the tissue-specific activity of tibolone and its metabolites. The ability of seven major expressed human sulfotransferase (SULT) isoforms to sulfate tibolone and its three metabolites was examined. Expressed human SULT2A1 was capable of sulfating tibolone and all three metabolites with the highest affinity for 3alpha-OH-tibolone. SULT1E1 conjugated both 3-OH-tibolone metabolites and tibolone itself slightly. SULT2B1b sulfated both 3-OH metabolites but not tibolone or Delta(4)-tibolone. SULT isoforms 1A1, 1A3, 1B1 and 1C1 did not demonstrate detectable activity. Sulfation of tibolone and its metabolites by human tissue cytosols was analyzed to determine whether the pattern of tibolone sulfation corresponded to the known expression of SULT isoforms in each tissue. The tissue-specific effects of tibolone may be regulated in part by the inactivation of tibolone and its metabolites by specific human SULT isoforms.

Regulation of MCF-7 breast cancer cell growth by beta-estradiol sulfation.

Estrogen stimulation is an important factor in human breast cancer cell growth and development. Metabolism of beta-estradiol (E2), the major endogenous human estrogen, is important in regulating both the level and activity of the hormone in breast tissues. Conjugation of E2 with a sulfonate moiety is an inactivation process since the sulfate ester formed by this reaction can not bind and activate the estrogen receptor. In human tissues including the breast, estrogen sulfotransferase (EST, SULT1E1) is responsible for high affinity E2 sulfation activity. EST is expressed in human mammary epithelial (HME) cells but not in most cultured breast cancer cell lines, including estrogen responsive MCF-7 cells. Stable expression of EST in MCF-7 cells at levels similar to those detected in HME cells significantly inhibits cell growth at physiologically relevant E2 concentrations. The mechanism of cell growth inhibition involves the abrogation of responses observed in growth factor expression in MCF-7 cells following E2 stimulation. MCF-7 cells expressing EST activity did not show a decrease in estrogen receptor-alpha levels, nor a characteristic increase in progesterone receptor or decrease in transforming growth factor-beta expression upon exposure to 100 pM or 1 nM E2. The lack of response in these MCF-7 cells is apparently due to the rapid sulfation and inactivation of free E2 by EST. These results suggest that loss of EST expression in the transformation of normal breast tissues to breast cancer may be an important factor in increasing the growth responsiveness of preneoplastic or tumor cells to estrogen stimulation.

Elevation of hepatic sulphotransferase activities in mice with resistance to cystic fibrosis.

The severity of intestinal disease in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) (-/-) mice has been reported to co-segregate with gene loci which contain the genes for hydroxysteroid sulphotransferase (SULT). Because of the potential involvement of steroid hormones in CF, we investigated levels of steroid SULT activity in the livers of CFTR mice to determine whether the levels of SULT activity correlate with the occurrence or severity of CF. To elucidate the possible role of SULT activity in ameliorating the deleterious effects of CF in CFTR (-/-) mice, we determined the levels of phenol SULT (PST), hydroxysteroid SULT [dehydroepiandrosterone (DHEA)-ST] and oestrogen SULT (EST) activity in control CFTR (+/+), heterozygous CFTR (+/-) and homozygous CFTR (-/-) mice, which survive to adulthood. The level of PST activity was not significantly different between any of the groups of mice, regardless of sex or genotype. Although DHEA-ST activity was significantly higher in female mice than in male mice, there was no difference in DHEA-ST activity that could be correlated with genotype. In contrast with PST and DHEA-ST activities, we found that some male and all female adult CFTR (-/-) mice had elevated, dramatically different levels of EST from both CFTR (+/+) and CFTR (+/-) mice. Results from these SULT activity experiments were confirmed by Northern-blot analysis of mouse-liver RNA. Subsequent studies with preweanling mice revealed no differences in the levels of EST that could be correlated with genotype. Thus this study indicates that EST is elevated significantly in CFTR (-/-) mice which survive to adulthood and provides important biochemical information that EST levels may be protective in CF.