Effect of pregnenolone-16 alpha-carbonitrile and dexamethasone on acetaminophen-induced hepatotoxicity in mice.

Recently, we demonstrated that a microsomal enzyme inducer with a steroidal structure, pregnenolone-16 alpha-carbonitrile (PCN), markedly decreased the hepatotoxicity of acetaminophen (AA) in hamsters. Therefore, it was of interest to determine if PCN, as well as another steroid microsomal enzyme inducer, dexamethasone (DEX), would decrease the toxicity of AA in mice, another species sensitive to AA hepatotoxicity. Mice were pretreated with PCN or DEX (100 and 75 mg/kg, ip, for 4 days, respectively) and were given AA (300-500 mg/kg, ip). Twenty-four hours after AA administration, liver injury was assessed by measuring serum activities of sorbitol dehydrogenase and alanine aminotransferase and by histopathological examination. Neither PCN nor DEX protected markedly against AA hepatotoxicity in mice; PCN tended to decrease AA-induced hepatotoxicity, whereas DEX was found to enhance AA-induced hepatotoxicity and it produced some hepatotoxicity itself. DEX decreased the glutathione concentration (36%) in liver and increased the biliary excretion of AA-GSH, which reflects the activation of AA, whereas PCN produced neither effect. Thus, whereas PCN has been shown to markedly decrease the hepatotoxicity of AA in hamsters, apparently by decreasing the isoform of P450 responsible for activating AA to N-acetyl-p-benzoquinoneimine, this does not occur in mice after induction with either PCN or DEX. In contrast, DEX enhances AA hepatotoxicity apparently by decreasing liver GSH levels and increasing the activation of AA to a cytotoxic metabolite.     

Sulfation of acetaminophen in isolated rat hepatocytes. Relationship to sulfate ion concentrations and intracellular levels of 3'-phosphoadenosine-5'-phosphosulfate

The relationships among sulfate ion concentration, rates of acetaminophen (APAP) sulfation, and intracellular levels of the cofactor for sulfation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) were examined in isolated rat hepatocytes. APAP sulfation rates increased as the sulfate ion concentration was raised to 1.0 mM, after which further increases in sulfate ion concentration failed to influence rates of sulfation. Cellular PAPS levels were directly related to the sulfate ion concentration both in the presence and absence of APAP. However, PAPS levels were reduced by as much as 93% in the presence of APAP. At sulfate ion concentrations below 1.0 mM, the dependence of both rates of sulfation and levels of PAPS on the availability of sulfate ion indicates that rates of sulfation may be limited by the availability of PAPS when sulfate ion concentrations are in the physiological range. Because higher sulfate ion concentrations (greater than 1.0 mM) increased intracellular concentrations of PAPS without producing corresponding increases in APAP sulfation, phenol sulfotransferase activity may be rate limiting in the presence of high concentrations of sulfate ion.     

Effects of molybdate and pentachlorophenol on the sulfation of acetaminophen

Pentachlorophenol (PCP) is an inhibitor of phenol-sulfotransferases and has been used to ascertain the role of sulfation in toxicology. Recently, molybdate has been shown to inhibit the sulfation of various chemicals by decreasing hepatic concentrations of the cosubstrate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The purpose of this study was to compare the effectiveness of these two chemicals in inhibiting the sulfation of various doses of acetaminophen (AA) in the rat. PCP (40 micromol/kg) decreased the 2-h combined biliary and urinary excretion of AA-sulfate by 78, 83, 84, and 47% of the 0.1, 0.3, 1, and 3 mmol/kg doses of AA, respectively. Molybdate (7.5 mmol/kg) decreased the sulfation of these same doses of AA by 50, 65, 62, and 81%, respectively. These data indicate that PCP is more effective in decreasing the sulfation of low than high doses of AA, which may result from less AA, at lower doses, to compete with PCP for sulfotransferases. Conversely, molybdate is more effective in decreasing sulfation of high rather than low doses of AA because molybdate decreases sulfate availability and decreases PAPS synthesis. More PAPS is required for the sulfation of high than low doses of AA. Therefore, PCP inhibits sulfation more effectively at low doses of AA when sulfation is limited by sulfotransferases, and molybdate inhibits sulfation more effectively at high doses of AA when sulfation is limited by PAPS.     

Effect of microsomal enzyme inducers on biliary and urinary excretion of acetaminophen metabolites in rats. Decreased hepatobiliary and increased hepatovascular transport of acetaminophen-glucuronide after microsomal enzyme induction

Treatment of rats with phenobarbital (PB), 3-methylcholanthrene, and pregnenolone-16 alpha-carbonitrile increased the total (biliary plus urinary) excretion of thioether and glucuronic acid conjugates of acetaminophen (AA) without influencing AA-sulfate excretion, suggesting that these microsomal enzyme inducers enhance both cytochrome P-450-mediated toxication and UDP-glucuronosyltransferase-mediated detoxication of AA. However, induction with transstilbene oxide (TSO) did not increase the total excretion of AA-thioethers or AA-glucuronide and decreased AA-sulfate excretion. In addition, all inducers increased the ratio of AA metabolites excreted into urine over that excreted into bile. The extent of this shift from biliary to urinary excretion was dependent on both the AA metabolite and the inducer. The largest shift in the excretory route was seen with AA-glucuronide and induction with PB and TSO as inducers. Specifically, PB and TSO treatments decreased biliary excretion of AA-glucuronide by 70 and 89%, respectively, and increased its blood concentration up to 6- and 11-fold and urinary excretion 3- and 3.6-fold, respectively. Galactosamine depletes UDP-glucuronic acid from the liver only, thereby inhibiting hepatic but not extrahepatic glucuronidation. Galactosamine treatment prevented the PB-induced increase in AA-glucuronide in blood and urine. This suggests that the PB-induced increases in AA-glucuronide in blood and urine originated from the liver. Thus, microsomal enzyme inducers not only influence xenobiotic biotransformation, but may also after the contribution of the excretory routes (i.e. bile and urine) in the elimination of xenobiotic metabolites by changing the direction of hepatic transport.     

UDP-glucuronosyltransferase activity toward digitoxigenin-monodigitoxoside. Differences in activation and induction properties in rat and mouse liver

Glucuronidation of digitoxigenin-monodigitoxoside (DT1), a metabolite of the cardiac glycoside digitoxin, is mediated by the microsomal isozymes, UDP-glucuronosyltransferase(s) (UDP-GT). The present studies examined the activation and induction properties of UDP-GT activity toward DT1 in hepatic microsomes of rats and mice. When compared to enzyme activity present in native (latent) microsomes of the rat (0.104 +/- 0.010 nmol/min/mg of protein), the activity toward digitoxigenin-monodigitoxoside in mouse native microsomes was 3.5-fold higher (0.379 + 0.44 mumol/min/mg of protein). After treatment with ionic (sodium cholate), zwitterionic [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)], or nonionic (Emulgen 911, Triton X-100) detergents, or with UDP-N-acetylglucosamine, enzyme activity in rat microsomes remained unchanged. In contrast, UDP-GT activity (DT1) in mouse liver microsomes treated with detergents or with the nucleotide was increased 2-3-fold above native enzyme activity. Pretreatment of rats with the microsomal enzyme inducers, 3-methylcholanthrene and phenobarbital, had no effect on this enzyme activity, whereas pretreatment with pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone (DEX) increased enzyme activity toward DT1 800 and 380%, respectively. These findings support the hypothesis that PCN and DEX induce a unique form of UDP-GT in the rat that selectively glucuronidates DT1. In marked contrast, the activity of this enzyme in mouse liver was not affected by pretreatment with any of the microsomal inducers, including PCN and DEX. In both rat and mouse, the P-450p-dependent N-ethylmorphine demethylase activity was increased 10-15-fold in PCN-pretreated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.     

Phenobarbital, beta-naphthoflavone, clofibrate, and pregnenolone-16alpha-carbonitrile do not affect hepatic thyroid hormone UDP-glucuronosyl transferase activity, and thyroid gland function in mice

The effects of representative liver enzyme inducers such as clofibrate (CLO), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and beta-naphthoflavone (NF) on hepatic microsomal thyroxin (T4)- UDP-glucuronosyl transferase (UGT) and triiodothyronine (T3)- UGT activities and thyroid function were evaluated in OF-1 male mice after a 14-day po administration. CLO, PB, and PCN induced histological liver hypertrophy, increases in liver weights, in microsomal protein and cytochrome P450 contents as well as increases in specific UGT activities. Despite this, no significant changes in T4-UGT and T3-UGT activities occurred after treatment by any of these compounds. Furthermore, no significant changes in serum T4 and T3 levels were observed and thyroid histology was not affected. NF treatment induced microvacuolation of hepatocytes but did not affect any of the other tested parameters. The results show that, in contrast to the widely described effects in rats, liver enzyme inducers do not affect hepatic thyroid hormone metabolism and thyroid function in mice, suggesting that this species should be less sensitive to thyroid tumor promotion by hepatic microsomal enzyme inducers than rats.     

Induction of T(4) UDP-GT activity,serum thyroid stimulating hormone,and thyroid follicular cell proliferation in mice treated with microsomal enzyme inducers

The microsomal enzyme inducers phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) are known to induce thyroxine (T(4)) glucuronidation and reduce serum T(4) concentrations in rats. Also, microsomal enzyme inducers that increase serum TSH (i.e., PB and PCN) also increase thyroid follicular cell proliferation in rats. Little is known about the effects of these microsomal enzyme inducers on T(4) glucuronidation, serum thyroid hormone concentrations, serum TSH, and thyroid gland growth in mice. Therefore, we tested the hypothesis that microsomal enzyme inducers induce T(4) UDP-GT activity, resulting in reduced serum T(4) concentrations, as well as increased serum TSH and thyroid follicular cell proliferation in mice. B6C3F male mice were fed a control diet or a diet containing PB (600, 1200, 1800, or 2400 ppm), PCN (250, 500, 1000, or 2000 ppm), 3MC (62.5, 125, 250, or 500 ppm), or PCB (10, 30, 100, or 300 ppm) for 21 days. All four inducers increased liver weight and hepatic microsomal UDP-GT activity toward chloramphenicol, alpha-naphthol, and T(4). PB and PCB decreased serum total T(4), but PCN and 3MC did not. Serum thyroid stimulating hormone was markedly increased by PCN and 3MC treatments, and slightly increased by PB and PCB treatments. All four microsomal enzyme inducers dramatically increased thyroid follicular cell proliferation in mice. The findings suggest that PB, PCN, 3MC, and PCB disrupt thyroid hormone homeostasis in mice.     

In vitro cytotoxicity of allyl alcohol and bromobenzene in a novel organ culture system

Two well-known hepatotoxicants, allyl alcohol (AA) and bromobenzene (BB), were studied using an in vitro system of cultured liver slices from control and phenobarbital-treated rats, respectively. Dose- and time-dependent increases in media lactate dehydrogenase (LDH), and decreases in slice K+ content and in protein synthesis were observed in rat liver slices incubated with either compound at concentrations between 0.1 and 1 mM over a period of 6 hr. The histopathological changes which occurred in the intoxicated slices appeared to parallel these biochemical changes. Additionally, the toxicity of either BB or AA, evaluated at 4 hr, was inhibited when slices were preincubated for 30 min with beta-ethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A) (0.1 mM) or pyrazole (1.0 mM), respectively. In this in vitro incubation system the cytotoxicity of xenobiotics can be studied under conditions where the multicellular hepatic lobular architecture is partially maintained, and alterations in biochemical and functional processes may be correlated to pathological changes.     

Histomorphological changes and cytochrome P450 isoforms expression and activities in precision-cut liver slices from neonatal rats

Precision-cut rat liver slices are a widely accepted in vitro tool for the examination of drug metabolism, enzyme induction or hepatotoxic effects of xenobiotics. After prolonged incubation, however, distinct histopathological changes and increasing losses in function are seen with liver slices from adult animals. Since tissue from neonatal animals is expected to be less vulnerable, in the present study liver slices from 1-day-old rats were examined for morphological changes and for the expression of different cytochrome P450 (CYP) isoforms after incubation for up to 24 h and after a 24 h in vitro exposure to beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or pregnenolone 16alpha-carbonitrile (PCN). In parallel, CYP activities were assessed by different model reactions in slice homogenates and in intact slices. Histopathological changes were less pronounced in liver slices from 1-day-old rats than in those from adult animals. During the 24 h of incubation even a maturation of the tissue occurred, since the proportion of haemopoietic stem cells declined and the glycogen content of the hepatocytes increased. The CYP expression pattern after 2 and 24 h of incubation was similar to that of normal liver specimens from neonatal rats showing a moderate CYP1A1, 2B1 and 3A2 expression. The immunostaining for CYP1A1 and 2B1 was elevated after incubation with BNF. PB enhanced CYP2B1 and 3A2 expression, and DEX and PCN increased CYP3A2 immunostaining. This induction pattern was paralleled by respective effects on the corresponding model reactions. Thus, besides increased viability, slices from neonatal rats are excellently suited for the evaluation of an in vitro induction of CYP enzymes as well.     

d-Cysteine as a selective precursor for inorganic sulfate in the rat in vivo: Effect of d-cysteine on the sulfation of harmol

d-Cysteine, the unphysiological isomer of the sulfur containing amino acid l-cysteine, is not utilized for protein synthesis, glutathione synthesis or taurine production; it was tested as a selective precursor for inorganic sulfate, required for sulfation of xenobiotics. Both cysteine isomers were injected intravenously in the rat, in order to investigate their sulfoxidation to inorganic sulfate. The rates of sulfoxidation were very similar, so that stereospecificity for the amino acid seemed not to play a role. When the rats were fed a low-protein diet (LP-diet; containing only 8% casein as source of amino acids) the serum sulfate concentration decreased to about 20% of control. Under these circumstances the rate of sulfoxidation of both isomers was decreased to the same extent. In order to confirm that both cysteine isomers were equally efficient in providing inorganic sulfate for sulfation of xenobiotics, a constant infusion of harmol (a substrate for sulfation) was given to rats fed the LP-diet. Administration of l- or d-cysteine yielded similar increases in sulfation of harmol under these conditions. These results show that d-cysteine can be used to selectively enhance sulfate availability.     

Metabolism of benzene in rat hepatocytes. Influence of inducers on phenol glucuronidation

Alterations of benzene metabolism in liver markedly influence benzene toxicity at extrahepatic target tissues. Therefore, generation of 11 phase I and II metabolites of benzene (including phenol, hydroquinone, catechol, benzene-1,2-dihydrodiol, their sulfates and glucuronides, and phenylglutathione) was compared in hepatocytes from 3-methylcholanthrene (MC)- or phenobarbital-treated rats and from untreated controls. At 0.1 mM benzene, total metabolism appeared to be unchanged by treatment with inducers. Phenylsulfate (35%), phenylglucuronide (15%), and phenylglutathione (12%) represented the major metabolites in hepatocytes from untreated controls. With hepatocytes from MC-treated rats, a pronounced shift from phenylsulfate to phenylglucuronide (increase to 34%) was observed, while the formation of unconjugated phenol, hydroquinone, and catechol was decreased (from 16 to 10%). A similar shift from sulfation to glucuronidation was seen in similar studies with phenol. Lineweaver-Burk analysis of microsomal phenol UDP-glucuronosyltransferase activity suggested that MC-treatment induced a high affinity isozyme (KM = 0.14 mM), in addition to the low affinity isozyme (KM = 3.1 mM) present in liver microsomes from untreated and phenobarbital-treated rats. It is concluded that induction by MC of a high affinity hepatic phenol UDP-glucuronosyltransferase effectively shifts benzene metabolism toward formation of less toxic metabolites. This shift may reduce toxic risks at extrahepatic target tissues.     

Rapid determination of rat hepatocyte mRNA induction potential using oligonucleotide probes for CYP1A1, 1A2, 3A and 4A1

1. A new assay to quantify mRNA levels in small numbers of rat hepatocytes has been developed for cytochrome P450 (CYP) isoforms 1A1, 1A2, 3A and 4A1. The assay uses sets of oligonucleotide probes end-labelled with [35S]-dATP to hybridize to mRNA in control- or drug-treated rat hepatocytes cultured on Cytostar-T 96-well scintillating microplates. 2. The rat hepatocyte induction potential (RHIP) assays for CYP3A, 1A1, 1A2 and 4A1 are sensitive and selective and have an excellent qualitative relationship with CYP induction data ex vivo. The robustness of the CYP3A assay was determined following a run of > 40 plates. The variation of the dexamethasone (DEX) response on each plate, calculated as %coefficient of variation, showed that there was no significant difference between the variability of the response to DEX. 3. Assay specificity for each CYP isoform was achieved by designing probes (four per isoform) antisense to coding regions of each CYP gene sequence. In the CYP3A RHIP assay, pregnenalone 16alpha-carbonitrile (PCN), DEX, clotrimazole (CLOT) and miconazole (MIC) were all good inducers of CYP3A mRNA; beta-napthoflavone (BNF) and methylclofenapate (MCP), however, did not induce CYP3A mRNA, further defining the specificity of this methodology. Specificity was similarly confirmed for the other CYP isoforms. 4. Ind50, the concentration of inducer required to elicit a 50% induction of CYP-specific mRNA, was derived for prototypical CYP inducers: BNF 0.54 and 0.17 microM (CYP1A1 and 1A2 respectively), 3-methylcholanthrene (3MC) 0.11 and 0.04 microM (CYP1A1 and 1A2 respectively), PCN 0.03 microM, DEX 0.17 microM, CLOT 0.48 microM, MIC 3 microM, TAO 3 microM (CYP3A), MCP 1.8 microM, clofibrate (CLOF) 65 microM and ciprofibrate (CIP) 1.9 microM (CYP4A1). Ind50 for BNF and 3MC at CYP1A2 was 3-fold lower than that at CYP1A1 indicating a subfamily difference in inducer potency. 5. Reducing the numbers of animals and the amount of compound required to study CYP induction is an important advantage of the RHIP assays over conventional evaluations in vivo. Typically four rats are dosed for 4 days using oral doses in the range 50-500 mg kg(-1) day(-1). In comparison, the amount of hepatocytes required to carry out all the studies reported herein may be obtained from a single animal (< 2 x 10(8) viable cells) and CYP induction investigated using microg rather than g quantities of drug substance. 6. With appropriately designed oligonucleotide probes, the RHIP technology can assess CYP induction in human hepatocytes, which together with preclinical data can contribute to improving the quality of compounds progressing into the expensive process of drug development.     

Influence of combined treatment with pregnenolone-16α-carbonitrile and spironolactone or phenobarbital on drug metabolism in rats

In female rats, pretreatment with pregnenolone-16α-carbonitrile (PCN), spironolactone or phenobarbital resulted in shorter zoxazolamine and hexobarbital sleeping times and more rapid plasma clearance of zoxazolamine and bishydroxycoumarin. All of these effects were slightly enhanced by simultaneous pretreatment with PCN and spironolactone and greatly increased by concomitant administration of PCN and phenobarbital. However, the observed increases in zoxazolamine and hexobarbital metabolism rates by submitochondrial liver fractions were not further augmented by pretreatment with both of the steroids, while the rates in animals treated with PCN and phenobarbital were only slightly greater than in those which received one of these inducers. It is suggested that PCN and spironolactone share a common receptor and induction mechanism, while the action of phenobarbital is slightly different.     

Effects of microsomal enzyme inducers upon UDP-glucuronic acid concentration and UDP-glucuronosyltransferase activity in the rat intestine and liver.

This study was conducted to evaluate UDP-glucuronosyl-transferase (UDP-GT) activity, UDP-glucuronic acid (UDP-GA) concentration, and UDP-glucose (UDPG) concentration in the rat intestine and liver following oral administration of butylated hydroxyanisole (BHA), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC), phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or trans-stilbene oxide (TSO). Microsomal UDP-GT activity was assayed in vitro with acetaminophen (AA), harmol (HA), and 1-naphthol (NA) as the aglycones. Intestinal HA and AA glucuronidation were enhanced by BHA, BaP, and TSO, whereas 3MC, PB, PCN, and TCDD augmented hepatic HA-glucuronide formation and BHA, PB, PCN, TCDD, and TSO significantly increased hepatic AA glucuronidation. All inducing agents except PB and PCN markedly increased both intestinal and hepatic NA glucuronidation. PB, PCN, and TCDD paradoxically decreased intestinal glucuronidation of AA and HA. A similar effect upon hepatic glucuronidation was not observed with any of the agents studied. Hepatic UDP-GA concentration was increased significantly by all inducers studied except PCN and TCDD, whereas hepatic UDPG concentration was increased only by BHA. In the intestine, significant increases in UDP-GA concentration were produced only by BHA and BaP, which also elevated intestinal UDPG. These results demonstrate that microsomal enzyme inducers evoke different effects upon intestinal and hepatic glucuronidation. These differences are manifested with regard to induced changes in UDP-GT activity as well as treatment-induced alterations in UDP-GA content. Thus, the present study further underscores the marked variance of intestinal and hepatic xenobiotic glucuronidation.     

Phenobarbital and dexamethasone induce expression of cytochrome P-450 genes from subfamilies IIB, IIC, and IIIA in mouse liver.

Phenobarbital (PB) and dexamethasone (DEX) induce several liver-specific cytochrome P-450 mRNAs in rats. We examined the induction of P-450 mRNAs from families IIB, IIC, and IIIA by PB and DEX in six inbred mouse strains. P-450IIB1-related mRNA species were induced 3- to 13-fold by PB and 3- to 15-fold by DEX in all animals. P-450IIC6-related mRNA species were induced 3- to 7-fold by PB in males and females, and up to 5-fold by DEX in most males but not in female mice, in which DEX was inactive. P-450IIIA-related mRNA species were induced 2- to 7-fold by PB and 2- to 20-fold by DEX in all animals of either sex. In DBA/2J female mice, both inducers triggered a comparable early response (4 hr) at a low dose (10 mg/kg) for all three gene subfamilies, the maximum being reached between 8 and 18 hr of treatment with 100 mg/kg. Under the optimal induction conditions, coadministration of PB and DEX did not lead to any further increase in the responses. These results demonstrate the existence of analogies, as well as striking differences in the inductive effects of PB and DEX between rats and mice. They also indicate the possible involvement of these inducers in related inductive pathways for three cytochrome P-450 gene subfamilies in mouse liver.     

Enantioselectivity of human hydroxysteroid sulfotransferase ST2A3 with naphthyl-1-ethanols.

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of several endogenous steroids and many hydrophobic xenobiotic alcohols. The substrate stereoselectivities of sulfotransferases may be critically important in determining their overall roles in metabolism of drugs, carcinogens, and other xenobiotics. In the present work, stereoselectivity of the human hydroxysteroid sulfotransferase ST2A3 (also variously named as SULT2A1 or human DHEA-ST) was examined through analysis of its catalytic activities with the enantiomers of 1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol. The kcat/Km value for sulfation of the R-(+)-enantiomer of 1-naphthyl-1-ethanol catalyzed by ST2A3 was 3.3 min-1mM-1, whereas the S-(-)-enantiomer was not a substrate for the enzyme. S-(-)-1-naphthyl-1-ethanol did however interact with ST2A3 as an inhibitor of the sulfation of dehydroepiandrosterone. This substrate stereospecificity was not present with the enantiomers of 2-naphthyl-1-ethanol, since both were substrates for the enzyme. Such differences between the sulfation of 1- and 2-naphthyl-1-ethanol are consistent with the importance of steric interactions between the ethanol group and a hydrogen atom at the peri-position (C8) on the naphthyl ring in 1-naphthyl-1-ethanol that combine with the topology of the enzyme's active site to determine stereospecificity.     

Influence of P-4502E1 induction on benzene metabolism in rat hepatocytes and on biliary metabolite excretion.

The influence of P-4502E1 induction on the metabolite pattern of benzene was studied in hepatocytes in vitro and in bile in vivo, and compared with that obtained with phenol (the major benzene metabolite). Eight metabolites from benzene and four from phenol (including conjugates) represented over 90% of total metabolites. Benzene metabolism (0.1 mM) in hepatocytes from isopropanol-treated rats (2.5 ml/kg, orally) was 3-fold higher than in corresponding cells from control rats, primarily because of increased formation of hydroquinone and phenylglutathione. Immunoblotting of microsomes revealed a parallel induction of P-4502E1 in hepatocytes from isopropanol-treated rats. In contrast, treatment with 3-methylcholanthrene or phenobarbital caused a decrease of P-4502E1, together with reduced benzene metabolism at 0.01 mM benzene. Addition of isoniazid (5 mM) resulted in a strong inhibition of benzene and phenol metabolism. Benzene metabolites were determined in bile following intraperitoneal administration of benzene (2.5 and 150 mg/kg). Biliary benzene metabolites were increased 2- to 3-fold after isopropanol treatment. Hydroquinone sulfate was identified as a major biliary metabolite of phenol. The results suggest that treatment with inducers of P-4502E1 leads, even at low benzene exposure, to an increased release of potentially myelotoxic metabolites from liver into the systemic circulation.