The formation of carbamazepine epoxide by rat liver microsomes: an investigation of the biphasic kinetic profile

A biphasic profile has been found for the rat hepatic microsomal formation of carbamazepine 10, 11 -epoxide from varying concentrations of carbamazepine (CBZ). The two optima for epoxide formation appeared at substrate concentrations of about 0.3 mM and 1.0 mM CBZ, respectively, with a nadir occuring at 0.4 – 0.6 mM CBZ. The biphasic nature of the velocity-substrate profile was not due to metabolism or disappearance of the epoxide. Pretreatment of rats with phenobarbital or CBZ produced an increase in the epoxide formation at both low and high CBZ concentrations, whereas phenytoin (DPH) pretreatment increased epoxide only at low CBZ concentrations. 3-Methylcholanthrene treatment did not increase epoxide formation at either low or high CBZ concentrations. High and low affinity processes for epoxide formation developed in parallel in young rats. DPH added $\text{in}$$\text{vitro}$ inhibited only the epoxide formation at high CBZ concentrations. This inhibitory effect increased with age of the rats. These findings indicate that CBZ 10, 11 -epoxide formation in rat liver microsomes proceeds by two metabolic pathways distinguished by substrate affinity and inhibition. Analysis of data from previous clinical studies reveals a biphasic pattern for plasma levels of CBZ, and its 10, 11 -epoxide.     

Effect of phenobarbital administration to rats on the level of the in vitro synthesis of cytochrome P-450 directed by total rat liver RNA.

Total liver RNA has been isolated from male rats at different time points subsequent to a single injection of phenobarbital, and the level of cytochrome P-450 synthesis directed by these RNA preparations in a cell-free translation system has been determined. It is observed that the maximum $\text{in vitro}$ synthesis of cytochrome P-450 occurs at 16 hours (3-fold above uninduced level) which is approximately 30 hours prior to the maximum induction of spectrophotometrically detectable cytochrome P-450 measured in liver homogenates. Thus, while cytochrome P-450 mRNA is involved in the induction process, its synthesis does not appear to be rate limiting. In addition, phenobarbital induced cytochrome P-450 is not synthesized $\text{in vitro}$ in a form larger than that isolated from endoplasmic reticulum, but rather is also found to have a molecular weight of 50,000.     

The degradation of different forms of cytochrome P-450 in vivo by fluroxene and allyl-iso-propylacetamide.

Treatment of uninduced, phenobarbital and 3-methylcholanthrene induced rats with fluroxene and allyl-$\text{iso}$-propylacetamide decreased hepatic microsomal cytochrome P-450 and equivalently decreased microsomal heme, aniline binding and $\text{p}$-nitroanisole demethylase. In contrast, ethylmorpnine demethylase, benzpyrene-3-hydroxylase and ethoxyresofurin deethylase were not in all cases decreased in proportion to the loss of cytochrome P-450. After phenobarbital induction fluroxene and allyl-$\text{iso}$-propylacetamide degrade multiple forms of cytochrome P-450, but degrade in the greatest amounts the form(s) of cytochrome P-450 inducible by phenobarbital. After 3-methylcholanthrene induction fluroxene preferentially degrades cytochrome P-448, while allyl-$\text{iso}$-propylacetamide is relatively specific for the form(s) of cytochrome P-450 inducible by phenobarbital.     

Comparison of aryl hydrocarbon hydroxylase and epoxide hydratase. Induction in primary fetal rat liver cell culture

The activity of aryl hydrocarbon hydroxylase (AHH) and/or epoxide hydratase (EH) is induced in primary fetal rat liver cell culture by benz[a]anthracene (BA), phenobarbital (PB), cigarette smoke condensate (CSC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and trans-stilbene oxide (TSO). The response of the two enzymes to the different chemicals varies as follows: (a) AHH is induced by lower concentrations of BA, PB and CSC than those required to significantly induce EH; (b) AHH is selectively induced by TCDD and by low BA concentrations; (c) the kinetics of AHH induction by BA, PB and CSC are faster than that of EH; (d) TSO is a selective inducer of EH. As described earlier for AHH, RNA and protein synthesis and the continuous presence of the inducer are required in the early phases of EH induction. Later, when the EH activity has reached a plateau, intact RNA and protein synthesis is not necessary to maintain the enzyme at its optimal value. The removal of the inducer determines a decay of the EH activity, allowing the estimation of a biological $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 72 h. TSO prevents the AHH induction by PB, but not that mediated by BA and CSC. Added together with PB, BA, CSC or PB plus BA, TSO induces the EH activity in a more than additive manner. This effect is only seen after 6 days of continuous treatment. These results indicate that in this tissue culture model, the mechanism of AHH and EH induction can clearly be dissociated.     

Synthesis of liver cytochrome P-450b in a cell-free protein synthesizing system

Live ppolysomes isolated from rats that had been treated with phenobarbital (PB) are able to incorporate [³H]leucine into total protein $\text{in}\text{vitro}$ at a rate almost five times that of polysomes prepared from control animals. Specific immunoprecipitation of translational products has shown that polysomes from induced animals synthesize cytochrome P-450b at a rate almost seven times greater than polysomes from control animals. The increased protein and cytochrome P-450b synthesis can be detected as early as 6 h following phenobarbital administration and reaches a maximum at 12–18 h. The results suggest that PB administration effects an increase in mRNA for cytochrome P-450b.     

The epoxide hydrolase catalyzed hydrolysis of trans-3-bromo-1,2-epoxycyclohexane. A direct proof for a general base catalyzed mechanism of the enzymatic hydration

The hydrolysis of (±)-$\text{trans}$-3-bromo-1,2-epoxycyclohexane in the presence of rabbit liver microsomes was investigated, and found to yield, beside $\text{c}$-3-bromocyclohexane-$\text{r}$-1,$\text{t}$-2-diol, 2,3-epoxycyclohexanol. It was demonstrated that the latter compound was the only product of the enzymatic reaction, whereas the diol resulted from a non enzymatic hydration in the reaction medium. These data provide the first direct proof for a general base catalysis in the enzymatic epoxide hydration, previously hypothesized on the basis of several lines of indirect evidence, and disprove alternative mechanisms involving protonation of the oxirane oxygen.     

Intracellular location of human fibroblast interferon messenger RNA

Human diploid fibroblast (FS-4) cells were induced to produce interferon mRNA by exposure to poly(rI)·poly(rC) plus cycloheximide. The intracellular location of interferon mRNA was investigated by differential centrifugation of the cytoplasm into a membrane (pellet) and a free (supernatant) fraction, followed by injection of mRNA isolated from either fraction into $\text{X}\text{.}\text{laevis}$ oocytes. When translation in FS-4 cells was prevented, most (85–90%) of the interferon mRNA activity was found in the free fraction. However, when translation was permitted, most (80–95%) of the interferon mRNA activity was found in the membrane fraction. These results are consistent with the predictions of the “signal hypothesis” (Blobel and Dobberstein, J. Cell Biol. 1975, $\text{67}$:835) for secretory proteins.     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

Stimulation of caffeine metabolism in the rat by 3-methylcholanthrene

Groups of adult male rats treated with 3-methylcholanthrene, phenobarbital or vehicles alone, were administered caffeine either orally or intravenously. Serum caffeine concentrations were measured by radioimmunoassay. In vehicle and phenobarbital pretreated animals, caffeine elimination kinetics were non-linear. In control animals, the $\text{in}$$\text{vivo}$ apparent Km was 8 μg·ml^?1 (40 μM) and the apparent Vmax was 0.1 μg·ml^?1·min^?1 (0.5 μM·min^?1). Phenobarbital pretreatment did not change the apparent Km but slightly increased the apparent Vmax. 3-Methylcholanthrene pretreatment dramatically altered the elimination kinetics of caffeine, whether caffeine was given orally or intravenously. The elimination of caffeine from serum of 3-methylcholanthrene pretreated rats was first order with a $\text{t}\text{1}\text{2}$ of approximately 14 minutes.Our results are consistent with the proposed involvement of the cytochromes P-450 monooxygenase system in the elimination of caffeine. In addition, our results suggest that caffeine is a moderately poor substrate for the cytochromes P-450 present in control and phenobarbital-pretreated rats, but a particularly good substrate for the form(s) induced by 3-methylcholanthrene.     

Sizing of two bacteriophage T4 specific messenger ribonucleic acids formed in vitro and in vivo

Messenger RNA for two T4 specific enzymes, deoxynucleotide kinase and α glucosyltransferase, have been sized by sedimentation on sucrose density gradients. The sedimentation constants of transferase and kinase mRNAs formed $\text{in vitro}$ were 21.5S and 14.5S respectively, regardless of the duration of incubation up to 20 min. Although the kinase mRNA isolated from cells infected with T4 phage for 10 min was the same size as found $\text{in vitro}$ (14.5S), the transferase mRNA was found in a segment approximating the size of the kinase mRNA (14.5S). The experiments indicate that α glucosyltransferase mRNA is formed first as a polycistronic message and is then processed to the smaller unit.     

Immunohistochemical localization of epoxide hydratase in rat liver

An antibody produced against epoxide hydratase (EC 4.2.1.63) which had been purified to apparent homogeneity from hepatic microsomes of phenobarbital pretreated rats was employed in an unlabeled antibody peroxidase-antiperoxidase technique to localize the enzyme at the light microscopic level in the livers of untreated rats. Immunohistochemical staining for epoxide hydratase was detected in parenchymal cells throughout the liver lobule. Cells within the centrilobular regions, however, were observed to be stained more intensely than were those within the midzonal and periportal regions of the lobule. The results of this immunohistochemical study thus demonstrate that epoxide hydratase does not exhibit a uniform pattern of distribution within the liver lobule in untreated rats.     

Hydroxylation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by rat liver microsomes

Ring hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur in the presence of liver microsomes prepared from both normal and phenobarbital induced rats. The metabolite was identified by mass spectrometry after selective extraction and purification by liquid chromatography. The microsomal catalyzed reaction was oxygen and NADPH dependent, inhibited by carbon monoxide and induced 4–5 fold by $\text{in vivo}$ phenobarbital pre-treatment. Phenobarbital induced microsomes hydroxylated the substrate at a rate of 17.6 nmoles/min/mg protein at 37°. A Type I difference spectrum was observed with phenobarbital induced microsomes that also displayed a substrate binding constant (K_s of 4 × 10^?5 M.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

Perinatal development of styrene monooxygenase and epoxide hydrolase in rat liver microsomes and nuclei

Nuclear enzymes were found to develop earlier than the corresponding microsomal activities. In fact styrene monooxygenase enzymatic activity at 18 days gestational age reached about half the values of adult animals, whereas fetal microsomal activity was only about $\text{1}\text{20}$ the adult level at the same age. In microsomes and nuclei the ontogenic development of epoxide hydrolase is slightly slower than styrene monooxygenase. This suggests that fetuses and newborn animals are exposed to higher risk of accumulation of styrene-7,8-oxide, a toxic and possibly teratogenic product of styrene monooxygenase.     

Glutathione peroxidase activity of glutathione-s-transferases purified from rat liver.

$\text{Rhizobium}$ hemeproteins P-450$\text{a, b}$, and $\text{c}$ cross react with antibodies to P-450_CAM and P-450_LM-2. Anti P-450_CAM IgG and phenobarbital, each bound to Sepharose 4B, were effective in purification of $\text{Rhizobium}$ P-450$\text{c}$; the latter was more convenient. The amino acid composition of highly purified $\text{Rhizobium}$ P-450$\text{c}$ resembles the compositions of P-450_CAM and P-450_LM-2. These results suggest that P-450 heme proteins of unrelated substrate specificities may nevertheless contain similar structural features.     

The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro.

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome $\text{b}$₅ or NADPH-cytochrome $\text{c}$ reductase $\text{in vitro}$.     

Comparison of ratios of covalent binding to total metabolism of the pulmonary toxin, 4-ipomeanol, In vitro in pulmonary and hepatic microsomes,and the effects of pretreatments with phenobarbital or 3-methylcholanthrene

Studies of the ratios of the amounts of 4-ipomeanol covalently bound to the total amounts metabolized support the view that the high rates of $\text{in}\text{vitro}$ pulmonary microsomal alkylation by 4-ipomeanol reflect high rates of NADPH-mediated metabolic activation of the compound rather than a relative deficiency of a microsomal detoxication pathway. Moreover, the ability of 3-methylcholanthene pretreatment, but not phenobarbital pretreatment, to shift the $\text{in}\text{vivo}$ target organ alkylation and toxicity of 4-ipomeanol from the lung to the liver in rats could not be explained by a major alteration in the balances between microsomal toxication and detoxication pathways measurable in the $\text{in}\text{vitro}$ systems examined, nor upon a major change in the nature of the reactive 4-ipomeanol metabolites produced in the lungs or livers of the pretreated animals.