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PubMed2006

Tandem mass spectrometry for the direct assay of enzymes in dried blood spots: application to newborn screening for mucopolysaccharidosis II (Hunter disease).

BACKGROUND: A treatment for mucopolysaccharidosis II (Hunter syndrome) has recently become available. Therefore, we developed a high-throughput assay method appropriate for newborn screening for the relevant enzyme, iduronate 2-sulfatase. METHODS: We synthesized a new iduronate 2-sulfatase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard. The assay uses a dried blood spot on a newborn screening card as the enzyme source. RESULTS: When the assay was tested on dried blood spots, the iduronate 2-sulfatase activity measured for 13 patients with Hunter syndrome was well below the interval found for 57 randomly chosen newborns. The assay was more sensitive than previously reported iduronate 2-sulfatase assays. CONCLUSIONS: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of Hunter syndrome. This method also has the potential to be carried out in multiplex fashion to assay several different enzymes relevant to lysosomal storage diseases that are assayed in a single infusion into the mass spectrometer.

Blood Specimen Collection↗
PubMed1990

A screening method for mucopolysaccharidoses with increased urinary excretion of sulfated N-acetylhexosamines.

Patients with mucopolysaccharidosis have been reported to excrete elevated amounts of sulfated N-acetylhexosamines in their urine. Based on this finding, a new and simple colorimetric screening test for these disorders is presented. Chromatography of whole urine on Dowex AG 1-X8, from each of 23 normal controls, 5 patients with mucopolysaccharidosis and one patient with multiple sulfatase deficiency, was used to separate the sulfated hexosamines. The fractions eluted with 2M NaCl were analyzed according to the method of Reissig. Patients with Sanfilippo syndrome, type A, Sanfilippo syndrome, type D, Maroteaux-Lamy syndrome, Morquio syndrome, type A, and multiple sulfatase deficiency were clearly distinguished from normal controls. The procedure appeared most sensitive for Sanfilippo syndrome, type D, and multiple sulfatase deficiency, each of which involves deficient activity of N-acetylglucosamine 6-sulfate sulfatase.

Chromatography, Ion Exchange↗
PubMed1978

Localization of lysosomal enzymes in retinal pigment epithelium of rats with inherited retinal dystrophy.

Four acid hydrolase activities are demonstrable by light microscopy in pigment epithelial cell lysosomes of rats (Royal College of Surgeons--RCS) with inherited retinal dystrophy and in control (Fischer) rats. The enzymes include acid phosphatase, aryl sulfatase, N-acetyl-beta-glucosaminidase, and esterase activities. No marked differences are observed in distribution or staining intensity of lysosomes in the two strains of rat. Acid hydrolase activities are not localized in sites other than lysosomes. Acid phosphatase and aryl sulfatase activities are also demonstrable by electron microscopy. In both strains, acid phosphatase reaction product is localized to various forms of lysosomes in pigment epithelial cells. A diffuse precipitate, considered to be nonspecific in origin, is seen in the cytoplasm, apical processes, outer segments (control), and outer segment debris (RCS). The precipitate is probably due to adsorption of lead from the incubation medium or of lead phosphate that diffuses from heavy accumulations in nearby lysosomes. Aryl sulfatase reaction product, in contrast to acid phosphatase, is localized to far fewer lysosomes and there is virtually no nonspecific precipitate. The findings indicate that lysosomes of RCS pigment epithelial cells possess several cytochemically demonstrable acid hydrolase activities. There is no evidence for the localization of acid phosphatase (or aryl sulfatase) activities in sites other than lysosomes.

Acid Phosphatase↗
PubMed1986

Steroid metabolizing enzymes associated with the microvillar membrane of human placenta.

17 beta-Hydroxysteroid oxidoreductase, as well as estrone sulfate and dehydroepiandrosterone sulfate sulfatases, were found in the plasma membrane of microvilli of the fetal syncytiotrophoblast. Because of their location, these enzymes may influence feto-maternal transfer of steroids circulating as sulfates, the utilization of sulfated estrogen precursors and the proportion of estrone and estradiol delivered towards fetal and maternal circulations. Microvillar vesicles isolated from human term placentas were disrupted in hypotonic medium to obtain a membrane preparation. A fraction of the estradiol 17 beta-oxidoreductase (E2DH) activity in the vesicle remained associated to the membrane after disruption and treatment with 2 M NaCl. The membrane-associated activity was resistant to inhibition with trypsin and did not react with a polyclonal antibody which neutralized cytosolic E2DH activity. The membrane-associated enzyme was solubilized with a cholate-glycerol buffer solution and purified on Sephadex G-100. The estimated molecular weight of the solubilized enzyme (137 kDa) appears to correspond to a tetramer since it was found to be about twice the size of the cytosolic enzyme. Both enzymes focused in polyacrylamide gels at pH 5.2. The Km relative to E2 of the membrane-associated E2DH (1.3 microM) differs from those of mitochondrial (0.43 microM), microsomal (0.69 microM) and cytosolic (11 microM) fractions. The cytosolic and the microvillar membrane associated 17 beta-hydroxysteroid oxidoreductases also differ in their specificity for C18 and C19 steroid substrates and in their pH dependence patterns. Sulfatases acting on estrone sulfate and dehydroepiandrosterone sulfate in microvillar membranes were insensitive to trypsin and as resistant to washes with 2 M NaCl as alkaline phosphatase. This data indicated that steroid sulfatases are also microvillar membrane associated enzymes of potential physiologic importance in the hydrolysis of estrogen precursors.

17-Hydroxysteroid Dehydrogenases↗
PubMed1978

Four cases of pregnancy with low estrogen production due to placental enzymatic deficiency.

4 cases of normal pregnancies associated with low estriol excretion and resulting in healthy infants are described, in which by in vitro studies on the placentas a steroid 3-sulfatase deficiency was found in 3 cases and a lack of activity of the aromatizing system was found in the 4th case. A reduced activity of the aromatizing system was also shown in one of the cases affected by steroid 3-sulfatase defect. The placental 3 beta-hydroxysteroid dehydrogenase, delta 4,5-isomerase system was measured in 3 cases and results were normal. The repetition of the specific lack of steroid 3-sulfatase was observed in 2 successive pregnancies of the same patient (case T.C.). It is interesting to note that, at variance with other reports, this patient delivered 2 fetuses, both female.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

Recognition of arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-phosphotransferase.

The critical step for sorting of lysosomal enzymes is the recognition by a Golgi-located phosphotransferase. The topogenic structure common to all lysosomal enzymes essential for this recognition is still not well defined, except that lysine residues seem to play a critical role. Here we have substituted surface-located lysine residues of lysosomal arylsulfatases A and B. In lysosomal arylsulfatase A only substitution of lysine residue 457 caused a reduction of phosphorylation to 33% and increased secretion of the mutant enzyme. In contrast to critical lysines in various other lysosomal enzymes, lysine 457 is not located in an unstructured loop region but in a helix. It is not strictly conserved among six homologous lysosomal sulfatases. Based on three-dimensional structure comparison, lysines 497 and 507 in arylsulfatase B are in a similar position as lysine 457 of arylsulfatase A. Also, the position of oligosaccharide side chains phosphorylated in arylsulfatase A is similar in arylsulfatase B. Despite the high degree of structural homology between these two sulfatases substitution of lysines 497 and 507 in arylsulfatase B has no effect on the sorting and phosphorylation of this sulfatase. Thus, highly homologous lysosomal arylsulfatases A and B did not develop a single conserved phosphotransferase recognition signal, demonstrating the high variability of this signal even in evolutionary closely related enzymes.

Animals↗
PubMed1990

Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl.

A female child of healthy, unrelated parents presented at 12 months of age with a history of moderately severe developmental delay, macrocephaly, dysmorphic facies, hypotonia, hepatosplenomegaly, mild generalized dysostosis multiplex, mucopolysacchariduria (dermatan and heparan sulfates), and Alder-Reilly bodies in peripheral blood leukocytes. Iduronate sulfatase activity in plasma was markedly depressed: 0.11 units/ml/h (normal, 1.75 +/- 0.56, N = 6). Analyses of arylsulfatases A, B, and C, heparan N-sulfatase, alpha-mannosidase, beta-mannosidase, beta-glucuronidase, beta-hexosaminidase, beta-galactosidase, and alpha-fucosidase activities in plasma, leukocytes, and/or cultured skin fibroblasts were all normal. Urinary sulfatide excretion was also within normal limits. Karyotypes of peripheral blood leukocytes and cultured skin fibroblasts were normal. Serum iduronate sulfatase activities in the parents were in the normal range (father, 1.63 units/ml/h; mother, 1.25 units/ml/h). The results of analyses of restriction fragment length polymorphisms (RFLP) of DNA from cultured skin fibroblasts with the use of probes for loci extending from Xpter to Xq28 showed X chromosome heterozygosity and confirmed the paternal origin of one of the X chromosomes. Studies on sulfur-35 uptake in mixed fibroblast cultures showed cross-correction of [35S]-glycosaminoglycan accumulation between cells from the patient and normal cells or cells from a patient with Hurler disease; however, there was no cross-correction between cells from the patient and those from boys affected with classical Hunter disease. This represents only the second confirmed case of Hunter disease reported in a karyotypically normal girl.

Arylsulfatases↗
PubMed1988

Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans.

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.

Animals↗
PubMed1986

Primary culture of cells from human chorion laeve: steroid metabolism and properties of cells grown in defined media supplemented with 0.1% or 10% fetal calf serum.

The purpose of this study was to develop primary cultures of human chorion laeve cells and examine certain aspects of steroid metabolism during culture. Tissues obtained by elective cesarean section at term (38-40 weeks) were dispersed with collagenase. Cells were isolated on Percoll gradients at the interface between 20% and 40% Percoll and examined in primary culture for up to 1 week. Cultures were carried out in chemically defined media supplemented with 10% or 0.1% fetal calf serum (FCS). The morphological and biochemical properties of the cells were different in the two systems. In 0.1% FCS, cells formed clumps of tissue within 16 h of plating, and there was no cell replication. In contrast, in 10% FCS, the cells formed a carpet of tissue and reached confluence after 5 days in culture, resulting in increased DNA and protein content and thymidine incorporation in the dishes. Three steroidogenic enzymes were studied during culture: alkyl steroid sulfatase, estrogen sulfatase and 3 beta-hydroxysteroid dehydrogenase. The sulfatases had higher activities in 0.1% than in 10% FCS, and their activities decreased markedly during the culture period. In contrast, 3 beta-hydroxysteroid dehydrogenase activity was higher in 10% FCS than in 0.1% FCS. Activity remained constant during the culture period in 0.1% FCS and increased in 10% FCS. In the latter system this increase resulted in the enzyme maintaining a constant specific activity during culture. These studies describe two viable systems of chorion laeve cells in primary culture, which may be valuable for studying long term and/or subtle effects on various metabolic aspects of this tissue.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

The human arylsulfatase-C isoenzymes: two distinct genes that escape from X inactivation.

Arylsulfatase C is a microsomal membrane-bound enzyme previously thought to be the same as steroid sulfatase, the only X-linked enzyme known to escape from X inactivation in man. We had shown that arylsulfatase C actually consists of two biochemically distinct isozymes, s and f. Only the s form has steroid sulfatase activity. The f and s forms were thought to be related through posttranslational or posttranscriptional modification of the same gene product. In part consistent with this hypothesis, we now report that in a panel of 28 rodent-human somatic cell hybrids, expression of both s and f was concordant only with the human X chromosome, thus showing that the f form is also X linked. In three separate somatic hybrids containing human X chromosomes in an inactive state, the f form was still expressed. Thus, similar to the s form, the f form also escapes from X inactivation. However, contrary to expectations, the s and f forms were not related by posttranslational modification of the same gene product. A full-length cDNA for the s form failed to hybridize to transcripts produced from an f-expressing cell line, showing that there is little sequence identity between the two. They are also not related by differential splicing of a common primary transcript, since fibroblasts from some patients with steroid sulfatase deficiency due to gene deletion of the s form continue to express the f form. Therefore, although the f and s isozymes of arylsulfatase C are X linked and escape from X inactivation, they are products from separate genes, thus providing a unique isoenzyme system to study possible gene duplication and regulation in the part of the human X chromosome that escapes inactivation.

Animals↗
PubMed1987

Phosphorylation of human lysosomal arylsulfatase B by cAMP-dependent protein kinase. Different sites of phosphorylation between normal and cancer tissues.

We previously demonstrated that an acidic variant (B1) of lysosomal arylsulfatase B from transplanted human lung cancer is phosphorylated on its protein and carbohydrate moieties (Gasa, S., and Makita, A. (1983) J. Biol. Chem. 258, 5034-5039). The present study identifies that a cAMP-dependent protein kinase is responsible for phosphorylation of arylsulfatase B. The protein kinase activity toward the sulfatase was considerably higher in the transplanted lung cancer than in normal lung in the presence of cAMP. B enzyme purified from normal human liver was found to contain 0.6 mol/mol B enzyme, and protein kinase treatment added further 1.3 mol of Pi to give a single phosphopeptide (X). On the other hand, B1 enzyme purified from the transplanted human lung cancer which had been labeled in vivo with 32Pi revealed at least two phosphopeptides (X and Y). Assuming that the sulfatase from normal liver and lung cancer possesses the same number of available phosphorylation sites, phosphorylation of site X which was available only by deliberate phosphorylation of the native, ordinary B enzyme appears to be cancer-associated. Increasing phosphorylation of the sulfatase resulted in a maximum 50% elevation in arylsulfatase activity, followed by a decrease of the activity upon overphosphorylation, using an artificial substrate.

Animals↗
PubMed1987

[Phosphorylation of lysosomal hydrolases in human cancer and its significance].

Many lysosomal hydrolases in cases of human cancer were found to be accompanied by acidic variant forms together with the major hydrolase components. Such variants were found to be phosphorylated not only at their carbohydrate moiety which contributes largely to their acidic property, but also at the protein moiety. We identified a cAMP-dependent protein kinase which is responsible for phosphorylation of arylsulfatase B. The protein kinase activity toward the sulfatase was considerably higher in transplanted lung cancer than in normal lung in the presence of cAMP. The B enzyme purified from normal human liver was found to contain 0.6mol of Pi/mol of B enzyme, and protein kinase treatment added a further 1.3mol Pi to give a single phosphopeptide (X) containing phosphothreonine. On the other hand, the B1 enzyme purified from transplanted human lung cancer which had been labeled in vivo with [32P] Pi revealed at least two phosphopeptides (X and Y). Assuming that the sulfatase from liver and lung cancer possesses the same number of available phosphorylation sites, phosphorylation of site X (Thr) which is available only by deliberate phosphorylation of the native, ordinary B enzyme, appears to be cancer-associated. Increased phosphorylation of the sulfatase resulted in a maximum 50% elevation in arylsulfatase activity, followed by a decrease in the activity upon overphosphorylation, using an artificial substrate.

Adenocarcinoma↗
PubMed1993

Capillary electrophoresis to measure sulfoesterase activity on chondroitin sulfate and heparin derived disaccharides.

Capillary electrophoresis was used to assay sulfoesterase activity on sulfated disaccharides derived from chondroitin sulfate, dermatan sulfate and heparin. The three sulfoesterases studied were chondro-4-O-sulfatase (EC 3.1.6.9) and chondro-6-O-sulfatase (EC 3.1.6.10) from Proteus vulgaris and heparo-2-O-sulfatase from Flavobacterium heparinum. Capillary electrophoresis was used to analyse sulfated disaccharide before and after sulfoesterase treatment and a change in migration time was indicative of the presence of sulfoesterase activity. This assay was used both on purified sulfoesterases and on minor sulfoesterase contaminants present in other enzyme preparations. The high sensitivity of capillary electrophoresis permits the elimination of 35S-radiolabeled substrates normally required to assay sulfoesterases. The high resolution of capillary electrophoresis allows the use of this assay on impure enzyme preparations containing high protein concentrations.

Chondro-4-Sulfatase↗
PubMed1999

Estradiol formation by human osteoblasts via multiple pathways: relation with osteoblast function.

The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.

17-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Sublobular distribution of transferases and hydrolases associated with glucuronide, sulfate and glutathione conjugation in human liver.

Activities of glucuronosyltransferase, sulfotransferase, glutathione S-transferase, beta-glucuronidase and sulfatase were determined in microdissected samples of periportal and pericentral sublobular regions from four human livers obtained at immediate autopsy. New methods are presented for the microdetermination of sulfotransferase and sulfatase activities in microdissected samples weighing 0.1 to 4 micrograms dry weight using umbelliferone and 4-methylumbelliferone sulfate as substrates. The three transferases were distributed heterogeneously across the liver lobule. Glucuronosyltransferase and glutathione S-transferase were localized predominantly in pericentral regions. In contrast, sulfotransferase activity was greater in periportal than pericentral regions. Average activities for glucuronosyltransferase and sulfotransferase were 23, and 50 mumoles X gm dry wt-1 X hr-1, respectively, in periportal regions, and 34 and 38 mumoles X gm dry st-1 X hr-1, respectively, in pericentral regions. Activities of glutathione S-transferase were considerably higher than those of the other transferases and were 8.3 mmoles X gm dry wt-1 X hr-1 in periportal areas and 12.2 mmoles X gm dry wt-1 hr-1 in pericentral areas. The two hydrolases studied, beta-glucuronidase and sulfatase, were evenly distributed across the liver lobule. The presence of significant hydrolase and transferase activities in both zones of the liver lobule supports the idea that net production of both sulfate and glucuronide conjugates may be influenced by futile cycling of conjugation-deconjugation reactions in both zones of the liver. Based on enhanced formation of sulfate but not glucuronide conjugates in homogenates of human liver treated with inhibitors of the hydrolases, it is suggested that futile cycling is more pertinent to the regulation of sulfation than glucuronidation.

Adolescent↗
PubMed2001

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes.

Cofactor-independent phosphoglycerate mutase (iPGM) has been previously identified as a member of the alkaline phosphatase (AlkP) superfamily of enzymes, based on the conservation of the predicted metal-binding residues. Structural alignment of iPGM with AlkP and cerebroside sulfatase confirmed that all these enzymes have a common core structure and revealed similarly located conserved Ser (in iPGM and AlkP) or Cys (in sulfatases) residues in their active sites. In AlkP, this Ser residue is phosphorylated during catalysis, whereas in sulfatases the active site Cys residues are modified to formylglycine and sulfatated. Similarly located Thr residue forms a phosphoenzyme intermediate in one more enzyme of the AlkP superfamily, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1 (autotaxin). Using structure-based sequence alignment, we identified homologous Ser, Thr, or Cys residues in other enzymes of the AlkP superfamily, such as phosphopentomutase, phosphoglycerol transferase, phosphonoacetate hydrolase, and GPI-anchoring enzymes (glycosylphosphatidylinositol phosphoethanolamine transferases) MCD4, GPI7, and GPI13. We predict that catalytical cycles of all the enzymes of AlkP superfamily include phosphoenzyme (or sulfoenzyme) intermediates.

Alkaline Phosphatase↗
PubMed1997

Distribution of cholesterol sulfate and its anabolic and catabolic enzymes in various rabbit tissues.

Cholesterol sulfate (CS) recently has been shown to be involved in signal transduction pathway. To evaluate its functional significance, we determined the concentration of CS, and the specific activities of cholesterol sulfotransferase and CS sulfatase in various tissues of rabbit, and compared them with the concentration of sulfoglycolipids in rabbit tissues. CS was present in the epithelia and mucosa, but not in the tunica muscularis, of the digestive tract, trachea, uterine endometrium and uterine cervix. It was also present in lung, spleen, kidney, prostate, skin, hair, and nail at relatively high concentrations. Its concentration in the uterine endometrium was nine times higher in pseudopregnant rabbits than in nonpregnant rabbits because of activation of cholesterol sulfotransferase and inhibition of CS sulfatase in the pseudopregnant rabbits. Sulfoglycolipids were not detected in the uterine endometria of either non-pregnant- or pseudopregnant rabbits. However, sulfoglycolipids were detected at relatively high concentrations in the cerebrum, cerebellum, stomach, duodenum, jejunum, testis, and kidney of rabbits and thus the tissues in which both sulfolipids were detected were the gastrointestinal tract and kidney. In the digestive tract, the concentration of CS decreased in the order esophagus, stomach, duodenum, and jejunum, but that of sulfatide increased in the same order, indicating distribution of CS in the squamous epithelium. In addition, both CS and sulfatide were detected in the serum. On the other hand, CS sulfatase activity was detected in all tissues examined, even in hair, from which the enzyme was liberated by brief sonication, and its highest specific activity was detected in the liver. The specific activity of cholesterol sulfotransferase varied among the tissues examined and was found to be significantly high in the esophageal epithelium and the uterine endometrium of pseudopregnant rabbit, indicating involvement of cholesterol sulfation in the formation of epithelium.

Animals↗
PubMed1994

Principal drug-metabolizing enzyme systems in L1210 leukemia sensitive or resistant to BCNU in vivo.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance has been mostly studied in vitro. In an attempt to better understand BCNU resistance in the in vivo situation, we compared the principal drug-metabolizing enzyme systems in two L1210 leukemia lines, one sensitive and one resistant to BCNU (L1210/BCNU), passaged in vivo in mice. The following enzymes were assayed by immunoblotting: cytochromes P-450 (1A1/1A2, 2B1/2B2, 2C8-10, 2E1, 3A), epoxide hydrolase (EH) and glutathione S-transferase (GST-alpha, -mu and -pi). The following enzymes and cofactors were assayed fluorometrically or spectrophotometrically: 1-chloro-2-4 dinitrobenzene-GST (CDNB-GST), total glutathione (GSH), UDP-glucuronosyltransferase, beta-glucuronidase, sulfatase and sulfotransferase. Results showed that cytochrome P-450 1A1/1A2 was the only isoenzyme detected in both L1210 and L1210/BCNU. CDNB-GST activity was significantly higher in L1210/BCNU compared with L1210. The isoenzyme GST-alpha was more abundant in L1210/BCNU compared with L1210, whereas GST-pi was expressed less in the BCNU-resistant leukemia line. GST-mu was not detected in either L1210 leukemia lines. GSH levels were similar in the two L1210 lines. No significant difference was observed between the two leukemia lines for the conjugative enzymes UDP-glucuronosyltransferase and sulfotransferase, whereas their corresponding hydrolytic enzymes beta-glucuronidase and sulfatase were about two-fold lower in the BCNU-resistant leukemia line. Epoxide hydrolase was 1.3-fold higher in L1210/BCNU compared with L1210 and this level was about three-fold higher than in mouse liver. In conclusion, these studies showed the presence of cytochrome P-450 1A1/1A2 in the two L1210 leukemia lines studied, and indicated noteworthy differences between the two leukemia lines for many enzyme systems such as GST, beta-glucuronidase, sulfatase and epoxide hydrolase. These data are of importance to better understand the mechanisms of drug resistance to nitrosoureas in vivo.

Animals↗