Biomedical subjects

J F Sinclair

Publications and source records attributed to J F Sinclair.

At least 37 records · Page 2Linked to original sources

PubMed1996

Carbon monoxide dehydrogenase from Clostridium thermoaceticum: quaternary structure, stoichiometry of its SDS-induced dissociation, and characterization of the faster-migrating form.

The molecular mass (M(r)) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300,000 +/- 30,000 Da. Since the enzyme is known to contain equal numbers of two types of subunits (M(r) = 82,000 Da for alpha and 73,000 Da for beta), this indicates an alpha 2 beta 2 quaternary structure. The enzyme was previously thought to have an alpha 3 beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent M(r) of about 420,000 Da. The disproportionately fast migration rate suggests that the enzyme is nonspherical. SDS induces the dissociation of an alpha subunit, yielding a stable species called FM-CODH. FM-CODH had a molecular mass of 210,000 +/- 30,000 Da, indicating an alpha 1 beta 2 structure. It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alpha 1 beta 2, exhibited S-->Fe charge-transfer transitions typical of Fe-S proteins, and afforded the gav = 1.82, 1.86, and 1.94 EPR signals. Quantitation of the 1.82 and (1.94 +/- 1.86) signals afforded 0.35 and 1.9 spin/alpha 1 beta 2, respectively. FM-CODH samples exhibited CO oxidation activity, but little CO/acetyl-CoA exchange activity. Some FM-CODH samples exhibited CO oxidation activities as high as native enzyme. These results, along with the quantified spin intensities of the EPR signals, indicate that FM-CODH contains the B- and C-clusters and suggest that these clusters are located in the beta subunit. The alpha subunit that dissociated during formation of FM-CODH is not required for CO oxidation activity. FM-CODH is either devoid of A-clusters, or if such clusters are present, they have lost their ability to exhibit substantial NiFeC signals and CO/acetyl-CoA exchange activity. Incubating FM-CODH and alpha yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an alpha 2 beta 2 structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible.

Aldehyde Oxidoreductases↗

PubMed1995

Acute hepatotoxicity of acetaminophen in rats treated with ethanol plus isopentanol.

Acetaminophen (APAP) hepatotoxicity was investigated in rats fed ethanol and isopentanol alone or in combination in a liquid diet for 7 days. Serum levels of aspartate aminotransferase (AST) and histological examination of liver slices were used to assess hepatotoxicity. At 7 hr after intragastric administration of 0.5 or 1.0 g APAP/kg, there was no significant increase in serum levels of AST in rats treated with APAP alone, or in rats pretreated with ethanol or isopentanol alone followed by APAP. There was mild central lobular congestion in the livers of rats pretreated with ethanol alone followed by APAP. In contrast, in rats pretreated with the combination of ethanol and isopentanol, administration of APAP caused a dramatic increase in serum levels of AST, along with marked central lobular necrosis, including steatosis and ischemic changes. Hepatic glutathione levels were decreased to 40-50% of control values in APAP-treated rats that had been pretreated with ethanol either alone or in combination with isopentanol. The serum concentrations of APAP were significantly lower in rats pretreated with the combination of ethanol and isopentanol followed by 1 g APAP/kg than in rats treated with APAP alone, suggesting a greater rate of APAP metabolism. We had reported previously that combined treatment of rats with ethanol and isopentanol resulted in additive to synergistic increases in CYP3A, with no further increases in CYP2E than that caused by ethanol alone. CYP3A may, therefore, be responsible for the increased APAP hepatotoxicity caused by the combined alcohol treatment.

Acetaminophen↗

PubMed1995

Ethanol and isopentanol increase CYP3A and CYP2E in primary cultures of human hepatocytes.

In primary cultures of human hepatocytes prepared from three separate livers, ethanol increased both CYP3A and CYP2E1, as detected immunochemically. Isopentanol, the major higher chain alcohol in alcoholic beverages, also induced CYP3A and CYP2E1. Maximal increases in these P450s occurred at the lowest concentrations of isopentanol examined, 0.1 mM. Ethanol and isopentanol were each more potent and more effective at inducing CYP3A in the human hepatocytes than was previously shown in cultured rat hepatocytes. Steady-state levels of CYP3A3/4 mRNA were increased by both ethanol and isopentanol. Ethanol and isopentanol induced immunoreactive CYP3A to a greater extent than did phenobarbital. In all three cultures, the increases in CYP3A after treatment with ethanol were less than those observed after treatment with rifampicin, a highly effective inducer of CYP3A in human hepatocytes. In one human hepatocyte culture, the lowest concentration of isopentanol tested increased CYP3A protein to an amount similar to that increased by rifampicin. In another human hepatocyte culture, however, the amount of immunoreactive CYP3A increased by isopentanol was less than that increased by rifampicin. In this latter culture, the steady-state levels of CYP3A3/4 mRNA increased by 0.1 mM isopentanol and 1 microM rifampicin were similar. This is the first finding of induction of CYP3A in human hepatocytes by ethanol or isopentanol. The clinical significance of the findings is discussed.

Adolescent↗

PubMed1995

Structure of bacterial luciferase.

The generation of light by living organisms such as fireflies, glow-worms, mushrooms, fish, or bacteria growing on decaying materials has been a subject of fascination throughout the ages, partly because it occurs without the need for high temperatures. The chemistry behind the numerous bioluminescent systems is quite varied, and the enzymes that catalyze the reactions, the luciferases, are a large and evolutionarily diverse group. The structure of the best understood of these intriguing enzymes, bacterial luciferase, has recently been determined, allowing discussion of features of the protein in structural terms for the first time.

Bacteria↗

PubMed1995

Ascorbic acid inhibits chemically induced uroporphyria in ascorbate-requiring rats.

Ascorbate was previously shown to suppress accumulation of uroporphyrin (URO) in cultured chick embryo hepatocytes and to competitively inhibit microsomal oxidation of uroporphyrinogen catalyzed by cytochrome P4501A2. Here we used the Osteogenic Disorder Shionogi (ODS) mutant rat, which cannot synthesize ascorbic acid, to examine the in vivo effect of ascorbic acid on hepatic URO accumulation caused by treatment with 3-methylcholanthrene (MC) and 5-aminolevulinate (ALA). Female mutant rats maintained on three levels of dietary ascorbate (15,200, and 800 ppm) were treated for a total of 24 days. On the 11th and 16th days, rats were administered 3-methylcholanthrene, and 5-aminolevulinate was present continuously in the drinking water from day 14. Hepatic URO accumulated at the two lowest ascorbate levels, but not at 800 ppm ascorbate. The latter dose produced normal hepatic ascorbate levels. Plasma ascorbate levels were proportional to the hepatic values. Male rats also accumulated URO at the low dietary dose of ascorbic acid. The methylcholanthrene-induced increase in microsomal levels of CYP1A1 and CYP1A2, total cytochrome P450, and activities of uroporphyrinogen oxidation and ethoxyresorufin deethylase were not affected by the dietary level of ascorbate. Neither male nor female Fischer 344 rats accumulated URO when treated with the MC/ALA regime. Hepatic ascorbate concentrations in these rats were five-fold to seven-fold higher than they were in mutant rats that developed uroporphyria on 150 ppm dietary ascorbate. In ODS rats fed ascorbate at 90 but not 900 ppm in the diet, hexachlorobenzene caused hepatic URO accumulation, indicating that the effect of ascorbic acid is not unique to the regimen using methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminolevulinic Acid↗

PubMed1994

Cocaine toxicity in cultured chicken hepatocytes: role of cytochrome P450.

Cocaine (COC) causes liver damage in several species, including man. Chicken embryo hepatocyte cultures were evaluated as a model system to investigate the mechanism of cocaine-mediated hepatotoxicity. Parameters used to assess toxicity were: (1) release of lactate dehydrogenase (LDH); (2) decreased induction of 5-aminolevulinic acid synthase (ALAS), measured as porphyrin accumulation; and (3) decreased protein synthesis. Exposure of untreated cultures to COC or norcocaine (NOR) caused dose-dependent increases in LDH release, decreased protein synthesis, and eventual cell death. Pretreatment with 2-propyl-2-isopropylacetamide (PIA), a phenobarbital-like inducer of cytochrome P450, accelerated toxicity and lowered the threshold dose at which toxicity occurred. PIA pretreatment also increased rates of elimination of both COC and NOR and increased rates of formation of NOR from COC. The toxicity of COC and NOR could also be detected as decreased porphyrin accumulation. Addition of the P450 inhibitor SKF-525A concurrently with COC or NOR decreased their rates of elimination. SKF-525A also prevented the increase in LDH release as well as the decrease in protein synthesis caused by treatment with COC or N-hydroxynorcocaine (N-OH). Addition of SKF-525A up to 3 hr after COC resulted in partial prevention of the LDH increase. Exposure of the cultures to COC induced cytochrome P450 2H protein. We conclude that this hepatocyte culture system is highly sensitive to COC toxicity and that constitutive as well as induced cytochrome P450 isoforms are involved in the production of liver damage from COC.

Allylisopropylacetamide↗

PubMed1994

Detection and localization of 3,3',4,4'-tetrachlorobiphenyl-induced P4501A protein in avian primary immune tissues.

P4501A can be detected in thymic and bursal microsomes from chickens pretreated with 3,3',4,4'-tetrachlorobiphenyl (TCB) using a polyclonal antibody against purified P4501A from 3-methylcholanthrene (3-MC)-induced chicken embryo liver. A dose-response for induction by TCB of P4501A protein was detected by Western blotting in both bursal and thymic microsomes. Ethoxyresorufin-O-deethylase (EROD), a specific catalytic activity of P4501A, was also induced in a dose-response fashion. More TCB-induced P4501A was detected in thymus than bursa by both methods. No EROD was detected in bursal or thymic microsomes from untreated chickens, although P4501A protein was detected at very low levels in thymic microsomes from untreated chickens. P4501A was detected by immunohistochemistry in scattered patches of non-lymphocytic cells residing in medullary regions of the TCB-induced thymus but was not detected in lymphocytes. This result supports previous work demonstrating that TCB-inducible EROD is much higher in the supporting tissue cell fractions than in lymphocyte fractions of the primary immune tissues. Although EROD was induced by TCB in the late stage embryo after 20 h exposure, no effect of TCB on the cell cycle in thymic or bursal lymphocytes was observed over the same period. The same TCB exposure resulted in bursal but not thymic cellular depletion. Thymic and bursal supporting tissue cells may be primary sites of immunosuppression within these organs by P4501A inducers or substrates whether immunosuppression occurs subsequent to metabolism or through interaction with Ah receptors.

Animals↗

PubMed1994

Kinetic partitioning during protein folding yields multiple native states.

The prevailing view in the field of protein folding holds that the native state is the most stable structure possible. A corollary of this thermodynamic hypothesis is that the native state is in equilibrium with all other conformations of the protein. We have found an example of a protein that may exist in two different states, both of which may be regarded as 'native', but which cannot equilibrate on a timescale that is biologically meaningful. We propose that the active conformation of this protein is at only one of several energy minima, and that during the process of refolding in vitro--and, we assume, folding in vivo--the choice of which state the polypeptide finally attains is determined by kinetic partitioning between folding pathways.

Kinetics↗

PubMed1994

Protoporphyrinogen accumulation in cultured hepatocytes treated with the diphenyl ether herbicide, acifluorfen.

Diphenyl ether (DPE) herbicides such as acifluorfen inhibit both the plant and mammalian forms of protoporphyrinogen oxidase, a heme biosynthetic enzyme. Only small amounts of protoporphyrin accumulated in primary cultures of chick embryo and rat hepatocytes treated with acifluorfen and the porphyrin precursor, 5-aminolevulinic acid. However, there was a large accumulation of the porphyrin precursor, protoporphyrinogen, which was detected after oxidation to protoporphyrin by an E. coli membrane enzyme. In contrast, conventional methods of porphyrin analysis which depend on quantitative autoxidation of protoporphyrinogen failed to detect this accumulation of protoporphyrinogen. This is the first demonstration that protoporphyrinogen can accumulate to high levels and remain stable in liver cells. In addition, we found that the effect of a protoporphyrinogen oxidase inhibitor such as acifluorfen on the regulation of heme synthesis in hepatocyte cultures differed from that of an iron chelator.

Aminolevulinic Acid↗

PubMed1994

Effects of hemopexin on heme-mediated repression of 5-aminolevulinate synthase and induction of heme oxygenase in cultured hepatocytes.

The serum protein hemopexin is considered to have a major role in the mechanism of the uptake of heme by hepatocytes by means of a heme-hemopexin receptor. Therefore, we examined in primary cultures of adult rat and embryonic chick hepatocytes whether the presence of hemopexin would affect the heme-mediated repression of 5-aminolevulinate synthase activity (the rate-limiting enzyme of heme biosynthesis) and the heme-induced increase of heme oxygenase activity (the rate-limiting step of heme degradation). Both of these heme-mediated effects were partly or entirely prevented by the presence of hemopexin. We conclude that homologous hemopexin, at molar concentrations exceeding that of heme, inhibited the uptake of heme into hepatocytes. These results suggest that heme, in amounts sufficient to affect the rate-limiting steps of heme synthesis and degradation, can only enter hepatocytes in primary culture when the binding capacity of hemopexin for heme has been exceeded or altered.

5-Aminolevulinate Synthetase↗

PubMed1994

Synergistic increases in rat hepatic cytochrome P450s by ethanol and isopentanol.

The purpose of this study was to determine if isopentanol alone or in combination with ethanol increased CYP2B1/2, CYP2E or CYP3A in the livers of rats. Increasing doses of isopentanol (0.5, 1, 2 or 3%) were administered in combination with 5.6% ethanol in the Lieber-DeCarli liquid diet for 7 days. Doses of 0.5 or 3% isopentanol were also administered alone. Isopentanol alone caused small increases in CYP2B1/2 and CYP3A. However, when isopentanol (2 or 3%) was combined with ethanol a synergistic increase in P4502B1/2 was observed. The combined alcohol treatment also resulted in a greater increase in immunoreactive CYP3A than either alcohol alone. Ethanol alone increased CYP2E 5-fold. Inclusion of isopentanol with ethanol resulted in either small or no additional increases in CYP2E. These results confirm our previous findings in cultured hepatocytes that when isopentanol is combined with ethanol, there is a synergistic increase in CYP2B1/2. Increases in CYP2B1/2, CYP2E and CYP3A protein moieties by ethanol, and by ethanol in combination with isopentanol, were associated with increases in their mRNAs. Blood isopentanol levels were 10-fold greater in rats administered 3% isopentanol in combination with ethanol compared to rats administered 3% isopentanol alone. From these results we suggest that isopentanol, a higher chain alcohol in alcoholic beverages, can contribute to increases in hepatic cytochrome P450 observed following consumption of alcoholic beverages.

Animals↗

PubMed1993

Ascorbic acid inhibition of cytochrome P450-catalyzed uroporphyrin accumulation.

Previous studies on the mechanism of the uroporphyria caused by polyhalogenated aromatic hydrocarbons have indicated a key role of cytochrome P450 of the 1A subfamily in catalyzing uroporphyrinogen (UROgen) oxidation. Here we report that ascorbic acid (ASC) inhibits UROgen oxidation in primary cultures of chick embryo hepatocytes and hepatic microsomes from chickens and mice. In hepatocyte cultures, 0.15 mM ASC totally prevented the accumulation of uroporphyrin (URO) induced by treatment of cells with the combination of 3,4,3',4'-tetrachlorobiphenyl (TCB) and 2-propyl-2-isopropylacetamide (PIA), but had no effect on the induction of protoporphyrin accumulation by PIA and desferrioxamine. However, addition of 5-aminolevulinic acid (ALA) to cultures treated with PIA plus TCB decreased the ability of ASC to prevent URO accumulation, suggesting that the effectiveness of ASC was dependent on the intracellular concentration of ALA or its metabolites. Similarly, when chick hepatocyte cultures were treated with TCB plus exogenous ALA to produce URO accumulation, the effectiveness of ASC was also less than when ALA was produced endogenously. Under this condition, addition of piperonyl butoxide, a P450 inhibitor, increased ASC inhibition of URO accumulation. ASC competitively inhibited the oxidation of UROgen by hepatic microsomes from chicks or mice treated with 3-methylcholanthrene (MC) with Ki for ASC being about 0.1 mM. ASC prevented formation of a 500-nm absorbing compound, probably tetrahydrouroporphyrin, the first intermediate in UROgen oxidation. These results are consistent with ASC preventing URO accumulation in hepatocytes by competitive inhibition of the first step of UROgen oxidation and suggest a new physiological role of ASC, that of maintaining UROgen in the reduced state.

Animals↗

PubMed1993

Folding of bacterial luciferase involves a non-native heterodimeric intermediate in equilibrium with the native enzyme and the unfolded subunits.

Bacterial luciferase is a heterodimeric enzyme that catalyzes the reaction of reduced FMN, O2 and an aliphatic aldehyde to yield the carboxylic acid and an excited flavin that emits blue-green light upon return to ground state. The two subunits of the luciferase from Vibrio harveyi, alpha and beta, have molecular weights of 40,108 and 36.349, respectively; the single active center resides primarily, if not exclusively, on the alpha subunit. We have found that bacterial luciferase can be unfolded in urea-containing 50 mM phosphate buffer, pH 7.0, and refolded by dilution of the urea with final luciferase concentrations of 5-25 micrograms/ml. We have analyzed the urea-induced equilibrium unfolding of bacterial luciferase by monitoring changes in both the far ultraviolet circular dichroism (predominantly secondary structure) and intrinsic fluorescence emission (predominantly tertiary structure) resulting from incubation in various concentrations of urea at 18 degrees C for 18-24 h. Both spectral methods indicated a biphasic unfolding transition; the first phase was protein concentration-independent, whereas the second phase was protein concentration-dependent. Equilibrium unfolding curves showed an increase in fluorescence up to 2 M urea followed by a decrease in intensity and red shift of the emission maximum. The ratio of the fluorescence emission in the presence of 2 M urea relative to that in the absence of urea was greater when fluorescence was excited at 295 nm than at 280 nm. The fluorescence increase in the 0-2 M urea range corresponded to the first phase of the biphasic unfolding process. The urea-induced loss of luciferase enzymatic activity appeared to correspond to the first transition observed with the spectroscopic methods, and likewise to be protein concentration-independent. These observations suggested a three-state unfolding mechanism in which the native heterodimeric enzyme rearranges to an inactive heterodimeric species that is well populated, followed by dissociation and unfolding of the alpha and beta subunits. The data were fit to a three-state mechanism using a nonlinear least squares method. At 18 degrees C in 50 mM phosphate, pH 7.0, the free energy change for the interconversion of the active heterodimer and the inactive heterodimeric species was estimated to be 4.52 +/- 0.30 kcal/mol; the free energy change for the interconversion of the inactive heterodimer and the individual subunits was 19.7 +/- 0.2 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular Dichroism↗

PubMed1993

Purified native subunits of bacterial luciferase are active in the bioluminescence reaction but fail to assemble into the alpha beta structure.

We have expressed the alpha and beta subunits of bacterial luciferase, encoded by luxA and luxB, from separate plasmids in Escherichia coli and developed an efficient purification scheme that yields many milligrams of protein of greater than 90% homogeneity. Earlier experiments showed that subunits synthesized separately assume conformations that do not assemble into the active luciferase heterodimer without prior denaturation. This observation led to the proposal that formation of the luciferase heterodimer involved interactions between intermediate conformations on the folding pathway of one or both of the subunits [Waddle, J. J., Johnston, T. C., & Baldwin, T. O. (1987) Biochemistry 26, 4917-4921]. Both of the subunits catalyze reduced flavin- and aldehyde-dependent bioluminescence reactions that are similar to that of the heterodimer in terms of reduced flavin binding affinity, aldehyde binding and inhibition, and kinetics of the overall bioluminescence reaction, but at an efficiency of about 5 x 10(-6) that of the heterodimer. Spectrophotometric analyses suggest that the structures of the individual subunits are similar to, but not identical to, the structures of the subunits in the heterodimer. Mixing of the two subunits under nondenaturing conditions did not lead to formation of the high specific activity heterodimer, even after prolonged incubation. Likewise, treatment of a stoichiometric mixture of the individual subunits with 5 M urea followed by 50-fold dilution of the urea did not yield the active heterodimer under the same conditions that yield high levels of active enzyme following denaturation of the native heterodimer [Ziegler, M. M., Goldberg, M. E., Chaffotte, A. F., & Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765].(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗

PubMed1993

Toxicity of paracetamol in cultured chick hepatocytes treated with methotrexate.

Cultured chick hepatocytes were used to investigate the hepatotoxicity of methotrexate alone and in combination with paracetamol. Treatment with methotrexate alone at concentrations as high as 1 mg/ml resulted in no toxicity in cultured chick hepatocytes, as indicated by no detachment of cells and no effect on protein synthesis or on release of the intracellular enzyme lactate dehydrogenase. However, treatment with methotrexate alone resulted in a 30% decrease in reduced glutathione levels. Combined treatment with methotrexate and paracetamol was toxic, but only in cells preinduced for cytochrome P450 1A by treatment with beta-naphthoflavone. Under these conditions, methotrexate lowered the threshold concentration of paracetamol at which toxicity was observed. This methotrexate-mediated increase in paracetamol toxicity was associated with decreased formation of the glucuronide, sulfate and thiol metabolites of paracetamol and with increased covalent binding of radiolabeled paracetamol to macromolecules. In cells pretreated with beta-naphthoflavone, additional treatment with either methotrexate or buthionine sulfoximine, an inhibitor of glutathione synthesis, together with paracetamol, was associated with decreased restoration of glutathione levels. These results suggest that methotrexate increased paracetamol toxicity by decreasing the amount of glutathione available for conjugation with reactive metabolites of paracetamol.

Acetaminophen↗

PubMed1993

Synergistic induction of cytochrome P450 by ethanol and isopentanol in cultures of chick embryo and rat hepatocytes.

The purpose of this study was to investigate whether isopentanol, the most abundant higher chain alcohol in alcoholic beverages, contributes to induction of different forms of hepatic P450s associated with consumption of these beverages. We have previously reported that ethanol and isopentanol each induce P450 2H1/2 in cultured chick hepatocytes and that ethanol induced P450 2B1/2, as well as P450 2E, in cultured rat hepatocytes. Here we investigated the induction of P450 by isopentanol alone and in combination with ethanol in cultured chick and rat hepatocytes. The forms of induced P450 were identified both enzymatically and immunochemically. In cultured chick hepatocytes, both isopentanol and ethanol induced P450 2E, but combined treatment with these alcohols had no greater effect than treatment with ethanol alone. In cultured rat hepatocytes, isopentanol alone did not induce P450 2E or 2B1/2 and had no effect on the ethanol-mediated induction of P450 2E. However, isopentanol combined with ethanol caused a synergistic induction of P450 2B1/2 in cultured rat hepatocytes and an additive to synergistic induction of P450 2H1/2 in cultured chick hepatocytes. The levels of enzyme activities induced by the combined alcohol treatment approached those induced by potent barbiturates. The results suggest that the pentanols in alcoholic beverages may be responsible for a barbiturate-like induction of P450 in hepatocytes.

Animals↗

PubMed1993

Norcocaine and N-hydroxynorcocaine formation in human liver microsomes: role of cytochrome P-450 3A4.

Cocaine was metabolized to norcocaine by microsomes prepared from lymphoblastoid cells expressing transfected human P-450 3A4. The specific activities of norcocaine formation by microsomes prepared from three human liver samples correlated with the amount of P-450 3A immunoreactive protein detected by immunoblot. Triacetyloleandomycin, a specific inhibitor of P-450 3A isoforms, inhibited formation of norcocaine from cocaine, but not formation of N-hydroxynorcocaine from norcocaine. The chemical identity of the norcocaine and N-hydroxynorcocaine produced by human liver microsomes was established by combination of gas chromatography and mass spectrometry. Thus, human P-450 3A4 is a cocaine demethylase, and P-450 isoforms of the 3A family are responsible for the majority of norcocaine production by human hepatic microsomes.

Cells, Cultured↗

PubMed1993

Dose-response relationships for the induction of P450 2B by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzne (TCPOBOP) in rat and cultured rat hepatocytes.

1. The dose-response relationships for hepatic CYP2B induction by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were examined in the male F344/NCr rat. TCPOBOP, administered for 14 days at 0-1000 ppm in the diet, caused concentration-dependent induction of hepatic CYP2B1 protein and RNA, and of CYP2B-mediated catalytic activities (benzyloxy- and pentoxyresorufin O-dealkylation, and testosterone 16 beta-hydroxylation). ED50 values for CYP2B induction were > or = 300 ppm dietary TCPOBOP. The maximal inductions observed were 66-88% of those resulting from exposure of the rats to 500 ppm dietary phenobarbital. 2. The EC50 values for hepatic CYP2B induction were 1.5-3.0 microM (based on serum TCPOBOP) and 15-20 mumol/kg liver. 3. The maximal inductions of isozymes of the CYP3A subfamily, of microsomal epoxide hydrolase, and of glutathione S-transferases Ya/Yc and Yb1/Yb2 in rats exposed to TCPOBOP were 58-74% of those resulting from exposure of the rats to 500 ppm dietary phenobarbital. 4. The ED50 value for induction of benzyloxyresorufin O-dealkylation in cultured rat hepatocytes by TCPOBOP was determined to be 0.93 microM. The maximal induction of this activity caused by TCPOBOP was 87% of the maximum increases caused by phenobarbital. 5. The results indicate that TCPOBOP is a highly effective phenobarbital-type inducer in the rat when administered in the diet for 2 weeks at 1000 ppm. When extent of induction is related to serum total xenobiotic level, TCPOBOP would appear to be at least as potent as, if not more potent than, phenobarbital in the rat.

Animals↗