Conjugation Capacity Research Articles

Sigma-class glutathione transferase from Xenopus laevis: molecular cloning, expression, and site-directed mutagenesis

The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined. Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues. The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST. To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys. The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity.

Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells

4-hydroxynonenal (4HNE) is a highly mutagenic and cytotoxic α,β-unsaturated aldehyde that can be produced in utero during transplacental exposure to prooxidant compounds. Cellular protection against 4HNE injury is provided by alcohol dehydrogenases (ADH), aldehyde reductases (ALRD), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). In the present study, we examined the comparative detoxification of 4HNE by aldehyde-metabolizing enzymes in a panel of adult and second-trimester prenatal liver tissues and report the toxicological ramifications of ontogenic 4HNE detoxification in vitro. The initial rates of 4HNE oxidation and reduction were two- to fivefold lower in prenatal liver subcellular fractions as compared to adult liver, and the rates of GST conjugation of 4HNE were not detectable in either prenatal or adult cytosolic fractions. GSH-affinity purification of hepatic cytosol yielded detectable and roughly equivalent rates of GST–4HNE conjugation for the two age groups. Consistent with the inefficient oxidative and reductive metabolism of 4HNE in prenatal liver, cytosolic fractions prepared from prenatal liver exhibited a decreased ability to protect against 4HNE-protein adduct formation relative to adults. Prenatal liver hematopoietic stem cells (HSC), which constitute a significant percentage of prenatal liver cell populations, exhibited ALDH activities toward 4HNE, but little reductive or conjugative capacity toward 4HNE through ALRD, ADH, and GST. Cultured HSC exposed to 5 μM 4HNE exhibited a loss in viability and readily formed one or more high molecular weight 4HNE-protein adduct(s). Collectively, our results indicate that second trimester prenatal liver has a lower ability to detoxify 4HNE relative to adults, and that the inefficient detoxification of 4HNE underlies an increased susceptibility to 4HNE injury in sensitive prenatal hepatic cell targets.

Developmental changes in glutathione S-transferase isoforms expression and activity in intrasplenic fetal liver tissue transplants in rats

The aim of the present study was to characterise developmental changes in glutathione S-transferase (GST) isoforms expression and in glutathione conjugation capacity in intrasplenic liver tissue transplants. For this purpose, syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fischer 344 rats. Three days, 1, 2, 4 weeks, 2, 4, 6 months and 1 year later, transplant-recipients and control animals were sacrificed and class alpha, mu and pi GST isoforms expression and GST activities using the substrates o-dinitrobenzene and 1-chloro-2,4-dinitrobenzene were assessed in livers and spleens. In the hepatocytes of the adult livers no class pi, but a distinct class alpha and mu GST expression was seen. The bile duct epithelia were class pi GST positive. Fetal livers displayed almost no class alpha and mu, but a slight class pi GST expression. The same pattern was seen in 3-day-old intrasplenic liver tissue transplants. Up to 2 weeks after surgery the class alpha and mu GST expression increased in the hepatocytes of the transplants, whereas the immunostaining for class pi GST disappeared. No remarkable changes were seen thereafter. Normal conjugation capacities were observed with the livers of both groups of rats. Control spleens displayed only low GST activities. From 2 months after transplantation on activities were significantly higher in transplant-containing spleens than in respective control organs with a further increase up to one year after grafting. These results show that intrasplenically transplanted fetal liver cells proliferate and differentiate into mature cells displaying a GST expression pattern with respective enzyme activities similar to adult liver.

Autistic disorder and gastrointestinal disease.

Autistic disorder is a pervasive developmental disorder manifested in the first 3 years of life by dysfunction in social interaction and communication. Many efforts have been made to explore the biologic basis of this disorder, but the etiology remains unknown. Recent publications describing upper gastrointestinal abnormalities and ileocolitis have focused attention on gastrointestinal function and morphology in these children. High prevalence of histologic abnormalities in the esophagus, stomach, small intestine and colon, and dysfunction of liver conjugation capacity and intestinal permeability were reported. Three surveys conducted in the United States described high prevalence of gastrointestinal symptoms in children with autistic disorder. Treatment of the digestive problems may have positive effects on their behavior.

Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis.

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359 Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

Pharmacokinetic analysis in serum of genistein administered subcutaneously to neonatal mice

Genistein, the principal soy isoflavone, was previously shown to induce uterine adenocarcinomas by 18 months of age in female CD-1 mice following administration by subcutaneous injections on postnatal days 1–5. A serum pharmacokinetic analysis of genistein in male and female mice treated identically showed that: maximal concentrations of total (conjugated+aglycone) genistein in females (6.8±1.4 μM) and males (3.8±1.1 μM, mean±SD) were comparable to those previously reported from dietary exposures in adult rats or in human infants consuming soy formulas; the average fraction present as active aglycone (31%) was similar to those in fetal and neonatal rats from placental and lactational exposures; and elimination half-times were longer than those in rats (three- to seven-fold) and adult humans (two- to three-fold). These results are consistent with a diminished capacity for enzymatic conjugation of genistein, based on the ontogeny, as an important determinant of estrogenicity in neonatal mice.

Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads ( Ameiurus nebulosus)

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads ( Ameiurus nebulosus) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S-transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-β estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p-nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward Δ5-androstene-3,17-dione (Δ 5-ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with male bullheads from Lake Woodruff. Collectively, our studies indicate the presence of reduced GST protein expression, reduced GST conjugative capacity and altered sex steroid homeostasis in female bullheads from a contaminated field site in Central Florida. The implications of these physiological alterations in terms of pollutant biotransformation and reproduction are discussed.

Trichloroethylene Risk Assessment: Relevance of Interindividual Differences

Interindividual variability in the disposition and effects of xenobiotics in humans and related inter-species differences should play a major role in human risk assessment. In particular for low-dose exposures to potentially carcinogenic compounds, novel tools and concepts are necessary to assess risks of chemical exposure, preferably based on susceptibility of sensitive individuals rather than on ‘average populations’. For trichloroethylene (TRI), significant controversy surrounds assessment of the carcinogenic potential associated with human exposure. Ample evidence exists that bioactivation of TRI via glutathione (GSH) S-transferase (GST)-mediated pathways, including the formation of chemically reactive S-containing intermediates by β-lyase in the kidney, is relevant for the mutagenic and carcinogenic potential of TRI. Using a novel, descriptive ‘bottom-up’ PBPK modeling approach, the relative and overall contribution of critical metabolic pathways can be predicted and compared between humans and laboratory animals. From these studies it is evident that humans: (a) show a minimal potency for GSH-conjugation of low-doses of TRI, (b) are in principle subject to large interindividual variations in this and in Cyt P450-dependent pathways of metabolism, and (c) show a much lower capacity for GSH conjugation of TRI than the rat. Taking these findings into consideration as well as the fact that kidney toxicity and kidney carcinogenicity of TRI are dose dependent and typically a feature of high and continuous exposure to TRI, it is unlikely that the nephrocarcinogenic potential of TRI observed in rat is a relevant human health hazard at currently reasonably foreseeable low levels of occupational and environmental exposure to TRI.

Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products.

Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (earlier designated as GST-2) is related to sigma class GSTs and was previously described as an indirect flight muscle-associated protein with no known catalytic properties. We now report that DmGSTS1-1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB), but has relatively high glutathione-conjugating activity for 4-hydroxynonenal (4-HNE), an electrophilic aldehyde derived from lipid peroxidation. 4-HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases. Drosophila strains carrying P-element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4-HNE. In flies with both, one, or none of the GstS1 alleles disrupted by P-element insertion, there is a linear correlation between DmGSTS1-1 protein content and 4-HNE-conjugating activity. This correlation indicates that in adult Drosophila 70 +/- 6% of the capacity to conjugate 4-HNE is attributable to DmGSTS1-1. The high abundance of DmGSTS1-1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress. Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S-transferases (e.g. GSTA4-4). The independent emergence of 4-HNE-conjugating activity in more than one branch of the glutathione S-transferase superfamily suggests that 4-HNE catabolism may be essential for aerobic life.

Glucose-6-Phosphate Dehydrogenase Deficiency: A Worldwide Potential Cause of Severe Neonatal Hyperbilirubinemia

After completing this article, readers should be able to: 1. Explain methods of preventing the hyperbilirubinemia caused by glucose-6-phosphate dehydrogenase (G6PD) deficiency. 2. Compare and contrast the correlation between hemolysis and serum bilirubin levels in healthy infants and those who are G6PD-deficient. 3. Describe the role of deficient bilirubin conjugation in the pathogenesis of G6PD deficiency-associated neonatal hyperbilirubinemia. 4. Define the risk for neonatal hyperbilirubinemia in female G6PD-deficient heterozygotes, G6PD-deficient homozygotes, and hemizygote males. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a commonly occurring X-linked genetic enzyme defect, is notorious for its association with acute hemolytic crises occurring in response to a frequently identifiable trigger (favism). Another potentially devastating danger of this condition is severe neonatal hyperbilirubinemia with its accompanying bilirubin encephalopathy, kernicterus, and death. Far from being a condition limited to historical time epochs and developing countries, G6PD deficiency-associated kernicterus still is seen in modern times. Indeed, among 80 infants from 21 states in the United States documented in a pilot Kernicterus Registry between 1984 and 1998, 18 (22.5%) were reported to have G6PD deficiency. Furthermore, Maisels recently listed G6PD deficiency among the 10 factors that are associated most commonly with an increased risk of nonhemolytic jaundice. Because of the association with favism, G6PD-associated hyperbilirubinemia traditionally has been regarded as hemolytic in origin. However, recent research has shown that although acute hemolysis does play a role, its contribution in many cases may be smaller than previously thought. The emphasis now has been placed on decreased bilirubin conjugation, with promoter polymorphism for the gene for the bilirubin conjugating enzyme, UDP glucuronoslytransferase 1A1 (UGT1A1), being a major factor in production of the icterus. In this review we highlight key aspects of the pathogenesis of this type of hyperbilirubinemia, explain the relevance of the condition, and describe a particularly problematic group that has a recently recognized high incidence of …

In vitro assessment of paracetamol-induced toxicity in the rat Reuber hepatoma H4IIEC3/G − cell line competent of xenobiotics metabolism

H4IIEC3/G − cells, descendants of rat Reuber hepatoma, were characterized for their response to paracetamol (acetaminophen; AAP) toxicity during log-phase of growth. The cells in culture were found to contain high contents of constitutive and dexamethasone inducible rat cytochrome P4503A besides other CYP members reported earlier. AAP produced dose-dependent decrease in cellular growth (50% at 0.7 m m). The drug steadily reduced the activity of UDP-glucuronyltransferase (UGT) towards 3-hydroxybenzo[ a]pyrene, decreased the contents of UDP-glucuronic acid (UDPGA) and significantly lowered GSH contents with length of exposure. After 48 hr of treatment, the GSH levels registered a fall of 50% while UGT and UDPGA exhibited a moderate decline of less than 25%. Decrease in the conjugation capacity of cells correlated with LDH leakage in the medium. Three compounds of natural origin, silymarin, kutkin and andrographolide at 10–20 μ m, offered relatively modest protection ranging from 24 to 55% at best, against the growth inhibitory effect of paracetamol. The hepatoma cells investigated appeared metabolically competent to respond to AAP toxicity and might prove useful for screening of agents against AAP-induced hepatotoxicity.

An evaluation of the carcinogenic potential of the herbicide alachlor to man.

Chronic bioassays have revealed that alachlor caused nasal, thyroid, and stomach tumours in rats but was not carcinogenic in mice. Significant increases in thyroid and stomach tumours were observed only at doses that exceeded the maximum tolerated dose (MTD). While nasal tumours were found at doses below the MTD, they were small and benign in nature. This publication describes the work undertaken by Monsanto to understand the carcinogenic mode of action of alachlor in the rat and to investigate the relevance to humans. The genetic toxicity of alachlor has been investigated in an extensive battery of in vitro and in vivo test systems. In addition, target-specific mutagenicity tests, such as the COMET assay and DNA binding in nasal tissue, were carried out to investigate any possible in-situ genotoxic action. The weight-of-evidence analysis of all available data clearly demonstrates that alachlor exerts its carcinogenicity in the rat by non-genotoxic mechanisms. In the rat, alachlor is initially metabolised primarily in the liver through the P-450 pathway and by glutathione conjugation. The glutathione conjugates and their metabolites undergo enterohepatic circulation with further metabolism in the gastrointestinal tract, liver, and then nasal tissue where they can be converted to a diethyliminoquinone metabolite (DEIQ). This electrophilic species binds to the cysteine moiety of proteins leading to cell damage and increased cell turnover. When comparisons of in vitro nasal metabolic capability were made, the rat's capacity to form DEIQ from precursor metabolites was 38 times greater than for the mouse, 30-fold higher than monkey, and 751 times greater than that of humans. This data is consistent with the results of studies showing in vivo formation of DEIQ-protein adducts in the nasal tissue of rats but not mice or monkeys. The lack of DEIQ nasal adducts in mice is consistent with the lack of nasal tumours in that species. When the differences between rat and humans in the capacity for initial glutathione conjugation by the liver and nasal tissue are also taken into account, the rat is found to be even more susceptible to DEIQ formation than man. Based on this, it is clear that the potential for DEIQ formation and nasal tumour development in humans is negligible. The mechanism of stomach tumour formation has been studied in the rat. The results demonstrated that the mechanism is threshold-sensitive and involves a combination of regenerative cell proliferation and a gastrin-induced tropic effect on enterochromaffin-like (ECL) cells and stem cells of the mucosal epithelium. The absence of a carcinogenic effect in mice and of any preneoplastic effect in monkeys treated with very high doses is indicative ofthe species-specific aspect of this mechanism of action. The results of studies on thyroid tumour production indicate that alachlor is acting indirectly through the pituitary-thyroid axis by increasing the excretion of T4 by enhanced glucuronidation and subsequent biliary excretion. The increased excretion reduces plasma T4 levels and a feedback mechanism leads to increased synthesis of TSH by the pituitary. Chronic stimulation of the follicular epithelium of the thyroid by TSH produces hyperplasia and ultimately tumour formation. This non-genotoxic, threshold-based mechanism is well established and widely considered to be not relevant to humans. In this work, the modes of action for the three types of tumours elicited in the rat by alachlor were investigated. All are based on non-genotoxic, threshold-sensitive processes. From all the data presented it can be concluded that the tumours detected in the rat are not relevant to man and that alachlor presents no significant cancer risk to humans. This conclusion is supported by the lack of mortality and tumours in an epidemiology study of alachlor manufacturing workers.

Enhancement of intestinal UDP-glucuronosyltranferase activity in partially hepatectomized rats

To evaluate whether a temporary hepatic insufficiency may affect intestinal glucuronidation, we determined UDP-glucuronosyltransferase activity towards bilirubin and p-nitrophenol in rat jejunum and liver after partial hepatectomy. Enzyme assays were performed in native, and in UDP- N-acetylglucosamine- or palmitoyl lysophosphatidylcholine-activated microsomes at different times post-hepatectomy. Content of enzyme was analyzed by Western blot. Microsomal cholesterol/phospholipid ratio, phospholipid and total fatty acid classes were also determined to evaluate the possible influence on enzyme activity. The results show that while hepatic microsomes exhibited no change in UDP-glucuronosyltransferase activity (for both substrates) with respect to shams at any time of study, intestinal activities increased significantly 48 h after surgery, returning to sham values 96-h post-hepatectomy. Western blotting confirmed the increase (about 50% for both substrates 48-h post-hepatectomy) in intestinal UDP-glucuronosyltransferase activity. No variations were observed in hepatic and intestinal microsomal lipid composition in agreement with the absence of modification in the percent of activation by palmitoyl lysophosphatidylcholine. In conclusion, jejunum but not liver, was able to produce a compensatory increase in conjugation capacity during a transitory loss of hepatic mass. The phenomenon is associated to a modification in the amount of UDP-glucuronosyltransferase, rather than to changes in the characteristics of the enzyme environment.

4-Substituted 1-chloro-2-nitrobenzenes: structure-activity relationships and extension of the substrate model of rat glutathione S-transferase 4-4.

In the present study, eleven 4-substituted 1-chloro-2-nitrobenzenes were tested for their GSH conjugation capacity when catalyzed by base or rat glutathione S-transferase (GST) 4-4. Kinetic parameters (ks and K(m), kcat, and kcat/K(m)) were determined and subsequently used for the description of structure-activity relationships (SAR's). For this purpose, eight physicochemical parameters (electronic, steric, and lipophilic) of the substituents and five computer-calculated parameters of the substrates (charge distributions and several energy values) were used in regression analyses with the kinetic parameters. The obtained SAR's are compared with corresponding SAR's for the GSH conjugation of 2-substituted 1-chloro-4-nitrobenzenes, previously determined [Van der Aar et al. (1996) Chem. Res. Toxicol. 9, 527-534]. The kinetic parameters of the 4-substituted 1-chloro-2-nitrobenzenes correlated well with the Hammett sigma p- constant; the Hammett sigma p constant corrected for "through resonance", while the corresponding kinetic parameters of the 2-substituted 1-chloro-4-nitrobenzenes did not. The base- and GST 4-4-catalyzed GSH conjugation reactions of 2-substituted 1-chloro-4-nitrobenzenes depend to a different extent on the electronic properties of the ortho substituents, suggesting the involvement of different rate-limiting transition states. The base- and GST 4-4-catalyzed conjugation of 4-substituted 1-chloro-2-nitrobenzenes, however, showed a similar dependence on the electronic properties of the para substituents, indicating that these substrates are conjugated to GSH via a similar transition state. Multiple regression analyses revealed that, besides electronic interactions, also steric and lipophilic restrictions appeared to play an important role in the GST 4-4-catalyzed GSH conjugation of 4-substituted 1-chloro-2-nitrobenzenes. Finally, the 4-substituted 1-chloro-2-nitrobenzenes were also used to extend the previously described substrate model for GST 4-4 [De Groot et al. (1995) Chem. Res. Toxicol. 8, 649-658], by which a specific steric restriction of substrates for GST 4-4 became clear.

Optimization of the hydrolysis of conjugated L-DOPA, dopamine and dihydroxyphenylacetic acid in human urine for assay by high-performance liquid chromatography with electrochemical detection.

Conjugates of the catechol compounds, L-dihydroxyphenylalanine (L-DOPA), dopamine and dihydroxyphenylacetic acid in human urine were analysed using the isocratic ion-pair reversed-phase HPLC method with electrochemical detection. Acid hydrolysis, using 4 mol/l HCl for 60 min, was more effective than treatment with sulphatase for the generation of free catechols. Free (non-conjugated) catechols already present, as well as those produced by either of the hydrolysis procedures, were adsorbed onto aluminium oxide and extracted in acid solution. The repeatability of the technique for within and between-batch urine analysis was less than 2% and 8%, respectively. Free urinary dopamine (and dihydroxyphenylacetic acid) concentrations were much higher in the urine of patients treated with L-DOPA for Parkinson's disease than in healthy volunteers. At high dopamine (and dihydroxyphenylacetic acid) levels the conjugation capacity was apparently exceeded, since the overall percent conjugation of L-DOPA, dopamine and dihydroxyphenylacetic acid was decreased "concentration dependently" where the concentrations of free catechols were increased. Both in the control group and L-DOPA-treated groups, enzymatic hydrolysis was much less effective than acid hydrolysis in generating free catechols. This indicated that there were other, non-sulphated conjugates in the urine, accounting for between 66 and 100% of total conjugates.

The effects of cyclic terminal groups in 4-aminoazobenzene and related azo dyes. Part 6. Electronic absorption spectra of some monoazo dyes derived from N-phenylisoindoline

Monoazo dyes containing a terminal isoindolinyl group absorb hypsochromically when compared with analogous compounds derived from N-phenylpyrrolidine due to the electronic influence of the benzene ring component of the heterocyclic substituent. The conjugative capacity of the isoindolinyl group is somewhat less than that of a pyrrolidinyl terminal ring.

Glutathione Conjugation of 1,2:3,4-Diepoxybutane in Human Liver and Rat and Mouse Liver and Lungin Vitro

1,3-Butadiene (BD) has been classified as a probable human carcinogen based on sufficient evidence of a carcinogenic response in B6C3F1mice and Sprague–Dawley rats and limited human evidence of carcinogenicity. Mice are much more susceptible to BD-induced carcinogenicity than rats. Previousin vitrostudies revealed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. BDE was also readily quantitated in blood and tissues of mice exposed to BD but could not be detected in rats similarly exposed. These findings suggest a key role for BDE in BD-induced carcinogenicity. The purpose of this study was to characterize the glutathione (GSH) conjugation of BDE by liver and lung cytosol from B6C3F1mice and Sprague–Dawley rats and human liver cytosol from six different individualsin vitro.BDE and glycine-[2-3H]GSH were incubated, at pH 7.4, with cytosol.13C NMR and mass spectral analysis indicated formation of two isomeric conjugates,S-(1-(hydroxymethyl)-2,3-epoxypropyl)glutathione andS-(2-hydroxy-3,4-epoxybutyl)glutathione, which were rapidly hydrolyzed in cytosol to the corresponding trihydroxy conjugates. Total conjugates were quantitated by HPLC. Conjugation of BDE with GSH followed Michaelis–Menten kinetics in human as well as rat and mouse cytosolic fractions. The conjugation rates in mouse and rat liver cytosol were similar (Vmax162 ± 16 and 186 ± 37 nmol/mg protein/min, respectively) and an order of magnitude higher than in human liver cytosol (Vmax6.4 ± 1.9 nmol/mg protein/min). The apparentKMvalues were lower in human (2.1 ± 1.4 mM) than mouse (6.4 ± 1.6 mM) or rat (24 ± 6 mM) liver. Mouse lung cytosol (Vmax38.5 ± 2.5 nmol/mg protein/min,KM1.70 ± 0.37 mM) is also more efficient in GSH conjugation than rat lung cytosol (Vmax17.1 ± 3.0 nmol/mg protein/min,KM4.2 ± 1.7 mM). These results suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in hepatic or pulmonary GSH conjugation of BDE. Also, considering the low oxidation rates of BD to BMO and of BMO to BDE in humans as compared to mice, the relatively low capacity of GSH conjugation of BDE in human liver will not necessarily lead to increased BDE blood levels in humans potentially exposed to BD.

Conjugation of Acrylonitrile and 2-Cyanoethylene Oxide with Hepatic Glutathione

The glutathione (GSH) conjugation of the rat carcinogen acrylonitrile (ACN) and its epoxide metabolite 2-cyanoethylene oxide (CEO) by rat, mouse, and human liver enzymes was characterized in vitro since GSH conjugation is the major disposition pathway for these chemicals in vivo. Mass spectral analyses indicated that S-(2-cyanoethyl)GSH was the product from reaction of GSH and ACN and that S-(cyanohydroxyethyl)GSH reaction products were formed from CEO. Because of the rapid nonenzymic reactions of ACN and CEO with GSH at pH 7.3, the steady-state kinetics of hepatic GSH conjugation were determined at pH 6.5 by HPLC analysis of the products. Hyperbolic kinetics were observed with respect to GSH for the reactions catalyzed by mouse or rat hepatic cytosols at pH 6.5, whereas sigmoidal kinetics were observed with respect to ACN or CEO. This kinetic pattern is consistent with the random sequential kinetic mechanism that has been described for GSH S-transferases. Estimates of the maximal velocities of the reaction at pH 6.5 showed that mouse enzymes had a 4- to 6-fold greater capacity for GSH conjugation of ACN and CEO than rat enzymes. ACN appeared to be conjugated with GSH more efficiently than CEO under these conditions. At physiological pH (7.3), rapid nonenzymic conjugation of GSH (10 mM) with ACN or CEO (5 mM) was observed (∼25 and 15 nmol product/min, respectively). Addition of hepatic cytosols or microsomes from rats or mice increased the velocity of GSH conjugation ∼1.6-fold. A similar velocity enhancement was observed with human liver cytosols for the GSH conjugation of ACN, but not for CEO. Human liver microsomes did not enhance the velocity of GSH conjugation of either substrate. These results suggest that ACN is a better substrate for human liver GSH S-transferases than CEO. Estimation of the initial velocities of the GSH conjugation reactions in intact rodent liver from the in vitro data at pH 7.3 suggests that the enzyme-mediated GSH conjugation of ACN and CEO will be ~4-fold greater than the velocity of the direct chemical reaction with GSH.

THE physiology of jaundice: Molecular and functional characterization of the Crigler-Najjar syndromes

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyltransferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 μM) CN type II patient A had 4.4 ± 2% residual activity and CN type II patient B had 38 ± 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with K m of 5.1 ± 0.9 μM and 7.9 ± 5.3 μM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, K m = 56 ± 23 μM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced. (J. Clin. Invest. 1994. 94:2385-2391.) Key words: hereditary diseases · site directed mutagenesis · enzyme-linked immunosorbent assay · enzyme kinetics · enzyme activity. (Reproduced from The Journal of Clinical Investigation, 1994, vol. 94, pp. 2385-2391 by copyright permission of The Society for Clinical Investigation.

Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase.

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyl-transferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 microM) CN type II patient A had 4.4 +/- 2% residual activity and CN type II patient B had 38 +/- 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 +/- 0.9 microM and 7.9 +/- 5.3 microM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 +/- 23 microM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced.