Hepatic Microsomal Heme Research Articles

Uroporphyrinogen III Synthase Knock-In Mice Have the Human Congenital Erythropoietic Porphyria Phenotype, Including the Characteristic Light-Induced Cutaneous Lesions

Congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error, results from the deficient but not absent activity of uroporphyrinogen III synthase (URO-synthase), the fourth enzyme in the heme biosynthetic pathway. The major clinical manifestations include severe anemia, erythrodontia, and disfiguring cutaneous involvement due to the accumulation of phototoxic porphyrin I isomers. Murine models of CEP could facilitate studies of disease pathogenesis and the evaluation of therapeutic endeavors. However, URO-synthase null mice were early embryonic lethals. Therefore, knock-in mice were generated with three missense mutations, C73R, V99A, and V99L, which had in vitro-expressed activities of 0.24%, 5.9%, and 14.8% of expressed wild-type activity, respectively. Homozygous mice for all three mutations were fetal lethals, except for mice homozygous for a spontaneous recombinant allele, V99A(T)/V99A(T), a head-to-tail concatemer of three V99A targeting constructs. Although V99A(T)/V99A(T) and C73R/V99A(T) mice had approximately 2% hepatic URO-synthase activity and normal hepatic microsomal heme and hemoprotein levels, they had 20% and 13% of wild-type activity in erythrocytes, respectively, which indicates that sufficient erythroid URO-synthase was present for fetal development and survival. Both murine genotypes showed marked porphyrin I isomer accumulation in erythrocytes, bone, tissues, and excreta and had fluorescent erythrodontia, hemolytic anemia with reticulocytosis and extramedullary erythropoiesis, and, notably, the characteristic light-induced cutaneous involvement. These mice provide insight into why CEP is an erythroid porphyria, and they should facilitate studies of the disease pathogenesis and therapeutic endeavors for CEP.

Concurrent induction of rat hepatic microsomal cytochrome P450 and haem oxygenase by 2,2'-dipyridyl ketone: comparison with the effect of 2,2'-dipyridyl amine

1. The effect of 2,2'-dipyridyl ketone and 2,2'-dipyridyl amine on the induction of hepatic microsomal cytochrome P450 (P450) and heme oxygenase was compared, and their effects on five different P450 isoforms (P4501A1, 3A2, 2B1, 2E1 and 2C11) in rat were examined. 2. Treatment of rat with 2,2-dipyridyl amine resulted in the marked induction of haem oxygenase to about seven-fold of the controls with a decrease in P450 content. 2,2-'Dipyridyl ketone produced concomitant induction of both P450 and haem oxygenase activity in a dose- and time-dependent manner without showing any sex differences. 3. Immunoblot analysis revealed that 2,2'-dipyridyl ketone slightly increased CYP2E1 and CYP3A2 at low doses, but not at high dose levels. There was no effect on P4502C11. P4502B1 was induced by the treatment with 2,2'-dipyridyl ketone in a dose-dependent manner. 4. These results indicate that dipyridyl compounds having different bridges between two aromatic moieties act as differential inducers of hepatic microsomal P450s and haem oxygenase.

Regio-Related Differential Induction of Hepatic Microsomal Heme Oxygenase and Cytochrome P450 by Substituted Imidazole and Pyridine Compounds in Rats.

The effects of variously subsituted pyridine and imidazole compounds and some of their chlorinated derivatives on the induction of hepatic microsomal heme oxygenase (HO) and cytochrome P450 (P450) were investigated in rats. 2-Benzylpyridine was able to induce HO to about 4.4-fold the control levels. 3-Benzylpyridine also increased HO to a lesser extent, but 4-benzylpyridine did not. Benzoylpyridines, phenylpyridines and diphenylmethylpyridines also produced a similar regio-differential induction of HO to benzylpyridines. Of the pyridine compounds examined, 2-phenylpyridine was the most potent inducer of HO. These substituted pyridines also increased the hepatic P450 content by inversely relating to the magnitudes of their abilities to induce HO. These results indicate that the substituted pyridines which have some lipophilic moieties, such as phenyl-, benzyl-, benzoyl- and diphenylmethyl-moieties, bound at the 2-position, but not at the 4-position of the pyridine ring, could induce HO. The substituted imidazoles generally produced a potent induction of P450, and some of them also exhibited HO induction similar to those of the corresponding pyridine compounds. Both 2-(4-chlorobenzyl)-pyridine and 2-(3, 4-dichlorobenzyl) imidazole were more potent inducers of HO than their dechlorinated parent compounds. All of these findings suggest that the substituted pyridine and imidazole compounds produce HO and P450 induction in a regio-related manner with lipophilic moieties, and that the addition of chlorine atom (s) may alter the magnitude of induction of HO in rat liver.

Heme oxygenase-1 mRNA levels, metallothionein mRNA levels, lipid peroxidation and microsomal CYP1A activities in rats treated with 3,3-dichlorobenzidine and some other inducers of P450.

The effect of 3,3-dichlorobenzidine (DCB), a potent inducer of CYP1A, on the levels of heme oxygenase-1 mRNA and metallothionein mRNAs was examined in the kidney, liver and lung of rats administered a single ip dose (157 μmol/kg) of the compound. DCB treatment increased heme oxygenase-I mRNA abundance in the kidney significantly from barely detectable levels in untreated animals; the maximum increase in the liver and lung was 24-fold and 4-fold, respectively. Hepatic microsomal heme oxygenase activity was also induced by DCB. In contrast with DCB, 2 other P450 inducers, β-naphthoflavone (β-NF) and phenobarbital did not elevate tissue HO-1 rnRNA levels. DCB pretreatment also elevated metallothionein mRNA levels in the kidney, liver and lung, with the effect in the lung being the least pronounced. In contrast with HO-1 mRNA, metallothionein mRNA was increased by the other P450 inducers examined. In vivo lipid peroxidation and in vitro NADPH-dependent microsomal lipid peroxidation were increased in the liver of DCB-treated rats but not in those of phenobarbital- or β-naphthoflavone-treated rats. Treatment with DCB or β-NF did not alter total hepatic microsomal P450 content, as measured spectrophotometrically, but induced the activity of CYP1A2. In contrast, the activity of CYP1A1 was induced to a lesser extent by DCB than by β-NF. The data show that DCB induces HO-1 as weD as P450 1A, confirm stimulation of lipid peroxidation by the compound, and suggest oxidative stress as a mechanism of HO-1 induction by the compound.

Differential effects of 3 dipyridyl isomers on hepatic microsomal cytochrome P450 and heme oxygenase in rats

We compared the effects of 3 dipyridyl isomers, 2,2′-dipyridyl, 2,4′-dipyridyl and 4,4′-dipyridyl, on hepatic microsomal heme oxygenase and drug-metabolizing enzyme activities in male rats. 2,2′-Dipyridyl increased cytochrome P450 (P450) content at lower doses, but decreased with increasing dose levels. Immunoblot analysis revealed that 2,2′-dipyridyl did not induce both P450 1A1/2 and P450 2B1/2, in contrast to 2,4′- and 4,4′-dipyridyls, both of which were inducers of either P450 1A1/2 and/or P450 2B1/2. Some drug-metabolizing enzyme activities gradually declined with the increasing dose level of 2,2′-dipyridyl. 2,2′-Dipyridyl was able to induce hepatic microsomal heme oxygenase in a dose-dependent manner, but 2,4′- and 4,4′-dipyridyls did not, even at the highest dose (0.80 mmol/kg) examined. Treatment of rats with 2,2′-dipyridyl resulted in the increase of glutathione (GSH) content in a dose-dependent manner, but not 4-substituted isomers. A time course study with 2,2′-dipyridyl revealed that it produced a significant decrease in hepatic GSH content at early time periods (2–6 h) after its administration with an inverse increase in heme oxygenase activity. The present investigation has revealed that in contrast to the induction of P450 by 4-substituted dipyridyl compounds, 2,2′-dipyridyl is a novel inducer of hepatic microsomal heme oxygenase, together with the change in hepatic GSH content. This study would provide information on the differential effects of simple dipyridyl isomers on hepatic enzymes involved in heme and drug metabolism.

14 EFFECT OF Sn- PROTOPORPHYRIN IN RATS WITH HYPERILURUBINEMIA

Sn-protoporphyrin is a synthetic metalloporphyrin that potently inhibits heme oxygenase, the rate limiting enzyme for heme degradation to bile pigment. Bile duct ligation producing cholestatis results in a marked increase in hepatic microsomal heme oxygenase.

The responses of hepatic monooxygenases of guinea pig to cadmium and nickel.

When Cd (3.58 mg CdCl2.H2O/kg, ip) was administered to male guinea pigs 72 h prior to sacrifice, the metal significantly inhibited the aniline 4-hydroxylase (AH) (16%), ethylmorphone N-demethylase (EMND) (26%), and aminopyrine N-demethylase (AMND) (18%) activities and cytochrome P-450 (12%) and cytochrome b5 (10%) levels. Cd did not alter the hepatic microsomal heme level. Cd, however, significantly increased the hepatic microsomal p-nitroanisole O-demethylase (p-NAOD) (53%) activity. When Ni (59.5 mg NiCl2 x 6H2O/kg, sc) was administered to the guinea pigs 16 h prior to sacrifice, the metal significantly depressed AH (49%), p-NAOD (66%), EMND (47%), and AMND (37%) activities, and cytochrome P-450 (15%), cytochrome b5 (24%), and microsomal heme (28%) levels. For the combined treatment, animals received the single dose of Ni 56 h after the single dose of Cd and then were killed 16 h later. In these animals, significant inhibitions were noted in AH (51%), EMND (47%), and AMND (30%) activities, and cytochrome P-450 (15%), cytochrome b5 (26%), and microsomal heme (30%) compared to those of controls. In the case of p-NAOD activity, the influence was in favor of Ni, i.e., the inhibition was about 61% by the combined treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cannabidiol-mediated cytochrome P450 inactivation

Cannibidiol (CBD) has been shown to impair hepatic drug metabolism in several animal species and to markedly inhibit mouse hepatic microsomal Δ 1-tetrahydrocannabinol (THC) metabolism by inactivating specific cytochrome P450s (P450) belonging to the 2C and 3A subfamilies. Elucidation of the mechanism of CBD-mediated P450 inhibition would be clinically very important for predicting its effect on metabolism of THC and the many other clinically important drugs known to be metabolized by P450s 2C and 3A. CBD-mediated inactivation of mouse hepatic microsomal P450s did not decrease hepatic microsomal heme content. However, [ 4C]CBD was found covalently bound to microsomal protein in an approximately equimolar ratio to P450 loss. Immunoprecipitation of microsomal protein with antibodies raised against either P450 2C or 3A revealed that approximately equal amounts of [ 14C]-CBD were bound to each of these P450s after CBD-mediated inactivation. Furthermore, this specific P450 binding was equivalent to P450 loss and accounted for nearly all of the microsomal [ 14C]CBD irreversible binding. Although >80% of the enzyme activities attributed to P450s 2C and 3A were inactivated by CBD at the anticonvulsant dose of 120 mg/kg, P450 2C was approximately 3-fold more sensitive than P450 3A at the lower CBD doses tested. CBD analogs were synthesized in order to elucidate the chemical pathways for CBD-mediated P450 inactivation in vivo. Oxidations at allylic carbon positions or saturation of either the exocyclic double bond or both double bonds of the terpene moiety did not markedly affect the inhibitory properties of the analogs. Methylation of both phenolic groups of the resorcinol moiety completely blocked the P450-inhibitory properties of this analog, revealing the involvement of a free hydroxyl group in the inactivation process. Rotation of the resorcinol moiety in abnormal-CBD did not impair the inhibitory properties of the analog, suggesting that the position of the hydroxyl group relative to the terpene ring is unimportant. Further studies are required to fully understand the chemical basis of CBD-mediated P450 inactivation.

Tin-protoporphyrin inhibits heme oxygenase and prevents the decline in hepatic heme and cytochrome P-450 contents produced in nude mice by tumor transplantation

Heme and hemeprotein perturbations are present in nude mice bearing transplanted tumors. Hepatic microsomal heme oxygenase activity is increased 50–100% in tumor bearing nu nu mice when compared with normal controls. This elevation in activity of the rate-limiting enzyme of heme degradation is associated with a 50% depletion of microsomal heme and cytochrome P-450 concentrations in liver. The synthetic heme analogue, Sn-protoporphyrin, a potent inhibitor of heme oxygenase, lowers the activity of heme oxygenase in tumor bearing animals to below control levels. This effect is associated with a normalization of hepatic heme and cytochrome P-450 contents. These findings might have implications for protecting normal cells during tumor growth and chemotherapy.

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in patients afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant ( K i ) for bilirubin was 1.5 μ m. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of δ-[4- 14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%,respectively, 72 h after biliary obstruction. The activities of hepatic δ-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%,respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of δ-aminolevulinic acid was not altered. During this 48- to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease States.

The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 μmol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [ 14C] CCl 4, spin-trapping of CCl 3 and CCl 4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe 2+, were also depressed in parellel with the decrease in microsomal enzyme activities.

Alterations in hepatic heme and cytochrome P-450 metabolism in murphy-sturm lymphosarcoma-bearing rats implications for drug metabolism

Previous studies have shown that tumor-bearing rats have significantly decreased hepatic microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity with, consequently, significantly decreased capacity for microsomal oxidative drug metabolism. Subsequent investigations have revealed that the rates of hepatic cytochrome P-450 apo-protein synthesis and degradation are decreased significantly and hepatic microsomal heme oxygenase activity is increased significantly in rats bearing an extra-hepatic tumor. Further studies have been done to attempt to clarify the pathogenesis and significance of these observations. Hepatic delta-aminolevulinic acid (ALA) synthetase activity in male Wistar rats declined to a nadir of 162 ± 34 (S.E.) pmoles ALA per mg protein per 30 min 6 days following i.m. transplantation of Murphy-Sturm lymphosarcoma (vs control = 218 ± 36 pmoles per mg per 30 min). Turnover of 3H-labeled heme in microsomal CO-binding particles (i.e. cytochrome P-450 heme) was increased significantly 8 days following i.m. transplantation of Murphy-Sturm lymphosarcoma with a T 1 2 of 5.5 hr for the fast phase of hepatic cytochrome P-450 heme disappearance in tumor-bearing rats as compared with a T 1 2 of 7 hr in control rats. Hepatic cytochrome P-450 apo-protein concentration was slightly, but not significantly, increased in Murphy-Sturm lymphosarcoma-bearing rats as compared with control rats up to 10 days following tumor transplantation. These results suggest that, in Murphy-Sturm lymphosarcoma-bearing rats, decreased microsomal cytochrome P-450 concentration is the result of both decreased cytochrome P-450 apo-protein synthesis and increased cytochrome P-450 heme turnover. Apo-cytochrome P-450 concentration was not appreciably altered because increased cytochrome P-450 heme turnover and decreased cytochrome P-450 apo-protein degradation were balanced by decreased cytochrome P-450 apo-protein synthesis. Because of their effects on cytochrome P-450 concentration and action, these alterations in heme and hemoprotein metabolism may be of importance in regulating oxidative drug metabolism in the tumor-bearing state.

Alterations of hepatic delta-aminolevulinic acid synthetase, heme oxygenase, microsomal cytochrome content and drug metabolism in rats bearing ascitic tumors AH 13, AH 66 and AH 414 and a 3-methylcholanthrene induced tumor.

Hepatic microsomal drug-metabolizing enzyme activities, cytochrome content, delta-aminolevulinic acid (ALA) synthetase and heme oxygenase activities were studied in rats bearing ascitic tumors AH 13, AH 66 and AH 414 and a primary, 3-methylcholanthrene (3-MC)-induced, tumor. Hepatic microsomal drug-metabolizing enzyme activities and cytochrome content were decreased in rats transplanted intraperitoneally with 1-2 x 10(6) cells of ascitic tumor cell lines AH 13, AH 66 and AH 414. The extent of the decrease of the microsomal cytochrome content and enzyme activities were dependent on the tumor-bearing periods after inoculation. Hepatic microsomal heme oxygenase activity was significantly increased concurrently with the decrease of microsomal drug-metabolizing enzyme activities and cytochrome content. Hepatic ALA synthetase was not changed appreciably in these tumor-bearing rats. Similar alterations of microsomal enzyme content and activities were observed in the livers of rats transplanted subcutaneously with AH 66 tumor cells and in rats bearing a primary tumor initiated by the subcutaneous injection of 3-MC. When the tumor was surgically removed from the rats bearing AH 66 subcutaneously, these hepatic microsomal parameters returned to normal levels. Microsomal drug-metabolizing enzyme activities and cytochrome content in these ascitic tumor cells were found to be at very low levels. From these results, if appears that there is an inverse relationship between the increase of microsomal heme oxygenase activity and the decrease of cytochrome P-450 and b5 as well as drug-metabolizing enzymes in the liver of tumor bearing rats.

Inhibition of uroporphyrinogen I synthase activity and depression of microsomal heme and cytochrome P-450 in rat liver by bilirubin

Purified rat hepatic uroporphyrinogen (UROgen) I synthase (URO-S) was inhibited by bilirubin or the ditaurine derivative. Inhibition was reversible and non-competitive to the substrate porphobilinogen (PBG). The inhibition constants (Ki values) for bilirubin and the conjugate were 1.5 μM and 0.26 μM respectively. Rats afflicted with hyperbilirubinemia caused by biliary obstruction had decreased levels of hepatic microsomal heme (58% of control) and cytochrome P-450 (60% of control) at day 3. Hepatic δ-aminolevulinic acid synthetase (ALAS) activity was increased (39% of control) at day 3.

Defective utilization of haem in selenium-deficient rat liver.

We have previously suggested that an inherent defect in hepatic haem utilization was responsible for the rapid stimulation of hepatic microsomal haem oxygenase activity observed in selenium-deficient rats given phenobarbital, a well known inducer of haem formation. To test this hypothesis, hepatic haem content was deliberately raised in selenium-deficient rats by administration of either tryptophan or allylisopropylacetamide, or by injecting haem itself. We now report that selenium-deficient rats are apparently relatively less efficient in utilizing hepatic haem than normal controls. The findings detailed in the present paper thus indicate that stimulation of hepatic microsomal haem oxygenase activity is indeed a manifestation of abnormal haem utilization in selenium deficiency. This suggests a novel role for selenium in hepatic haem metabolism.

Effect of Cholestasis Produced by Bile Duct Ligation on Hepatic Heme and Hemoprotein Metabolism in Rats

Cholestasis produced by bile duct ligation has been associated with decreased concentrations of hepatic microsomal cytochrome P450 and decreased hepatic microsomal oxidative drug metabolism. Bile duct ligation producing cholestasis results in a marked increase in hepatic microsomal heme oxygenase activity, with corresponding decreases in hepatic microsomal cytochrome P450 concentration, reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, and hepatic delta-aminolevulinic acid synthetase activity. As sham-operated rats also demonstrate a less prolonged decrease in cytochrome P450 concentration and reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, the metabolic effects of surgery and anesthesia must also be involved in these alterations in microsomal oxidative drug metabolism. The relative rate of hepatic cytochrome P450 synthesis and of degradation are both decreased after bile duct ligation. These data suggest that decreased hepatic microsomal cytochrome P450 concentrations in cholestasis are partly the result of decreased cytochrome P450 synthesis. Increased levels of heme oxygenase activity are not related to increased cytochrome P450 turnover, but may instead reflect enlargement and increased catabolism of a free heme pool resulting from decreased hemoprotein (cytochrome P450) synthesis.

Maternal β-Adrenergic Tocolysis and Neonatal Bilirubin Production

The potential for beta-adrenergic drugs to increase total bilirubin formation via cyclic adenosine monophosphate-mediated stimulation of hepatic microsomal heme oxygenase in the human neonate was evaluated. The pulmonary excretion rate of endogenously produced carbon monoxide (VeCO), an index of total bilirubin formation (TBF), was measured in 18 preterm neonates whose mothers received beta-adrenergic drugs for tocolysis and in 18 preterm neonates whose mothers were untreated. The mean VeCO of the neonates in the former group (17.2 +/- 7.3 microL/kg/hr) was the same as that in the latter group (17.4 +/- 6.2 microL/kg/hr); both values were elevated when compared with the mean VeCO of 20 term newborns (13.9 +/- 3.5 microL/kg/hr). Our findings indicate that TBF is not significantly increased in neonates whose mothers received beta-adrenergic drugs before delivery.

Biochemical basis for impaired drug metabolism in tumor-bearing rats: Evidence for altered regulation of hepatic microsomal hemeprotein synthesis

The pharmacologie effects of many drugs are enhanced in animals bearing tumors. This apparently stems from a decrease in the microsomal metabolism of these compounds in liver cells, owing to a decrease in either the activities of certain enzymes or in the content of cytochrome P-450 in microsomes, or both. Since impaired drug metabolism may have a direct bearing on the outcome of chemotherapy, a study of the biochemical basis for this alteration was begun. In female Sprague-Dawley rats bearing Walker 256 solid tumors i.m., pentobarbital metabolism was impaired as judged from prolonged sleeping-times. This effect was accompanied by a decrease in the microsomal content of cytochrome P-450. Analysis of hepatic microsomes by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that a microsomal protein of ~53,000 daltons was diminished in livers of tumor-bearing animals, when compared to normal controls, as determined by staining with Coomassie Blue. When rats were injected with [ 3H]leucine, although the dpm/100 μg of microsomal protein were the same for both normal and tumor-bearing rats, ~40% less 3H was associated with the microsomal proteins in the cytochrome P-450 region (~43,000 to ~58,000 daltons) of tumor-bearing animals than with those of normal controls. This was attributed to a decrease in the rate of synthesis of these, but not of other, microsomal proteins. Either staining of microsomal proteins with 3,3',5,5'-tetramethyl-benzidine, or labelling of them with δ-[ 14C]aminolevulinic acid, revealed that the overall content of microsomal hemeproteins of tumor-bearing rats was reduced considerably, when compared to normal controls (~70% as judged by the incorporation of 14C). These observations prompted investigations of the apparent rate-limiting synthetic and degradative enzymes of heme. We found that in the livers of 7-day tumor-bearing rats, the activity of δ-aminolevulinic acid synthetase was only 16% of control activity; conversely, the activity of hepatic microsomal heme oxygenase in the tumor-bearing rats was nearly eight-times greater than that of the normal animals. Together, these data indicate that perturbations in heme- and hemeprotein-synthesis cause the reduced content of cytochrome P-450 seen in tumor-bearing rats, and they provide a partial explanation for diminished drug metabolism by hepatic microsomes in the presence of a growing, transplantable, non-hepatic tumor.

Effect of honeybee ( apis mellifera) venom on the course of adjuvant-induced arthritis and depression of drug metabolism in the rat

The ability of honeybee venom to suppress Mycobacterium butyricum-induced arthritis was studied in Lewis rats. Bee venom, 2mg · kg −1 · day −1 for 24 days, suppressed but did not abolish the primary and secondary inflammatory responses to the adjuvant as monitored by decreases in the swelling of the left and right hind paws respectively. Bee venom itself caused no swelling of the hind paws. The effects of bee venom and adjuvant-induced arthritis on heme metabolism were also examined. Bee venom or adjuvant had no effect on hepatic δ-aminolevulinic acid synthase, porphyrin content, or ferrochelatase activity. However, with both treatments cytochrome P-450 and the associated enzymic activities of ethylmorphine N-demethylase and benzo[ a]pyrene hydroxylase were depressed markedly. In contrast, both treatments caused several-fold enhancement of hepatic microsomal heme oxygenase activity. Adjuvant-treated rats receiving bee venom showed changes in heme metabolism which were of a magnitude similar to those observed when either agent was administered to the experimental animals. Although the bee venom appears to suppress adjuvant-induced arthritis to a greater extent in female than in male rats, the alterations in heme metabolism were similar in bee venom-treated male and female rats. The observed changes in heme metabolism elicited by the venom or by the adjuvant are strongly suggestive of perturbations of the immune system causing alterations in hepatic microsomal enzymes.

Differential responses to inducers of delta-aminolaevulinate synthase and haem oxygenase during pregnancy.

The responses of hepatic delta-aminolaevulinate synthase and microsomal haem oxygenase to inducers were examined in pregnant rats. 2-Allyl-2-isopropylacetamide-mediated induction of delta-aminolaevulinate synthase was greatly decreased during pregnancy and in the early post-partum period. Administration of allylisopropylacetamide to pseudopregnant rats induced delta-aminolaevulinate synthase normally. Treatment of pregnant rats with cortisol failed to restore the drug-mediated induction of delta-aminolaevulinate synthase. Microsomal cytochrome P-450 content and the activities of drug-metabolizing enzymes such as aniline hydroxylase and ethylmorphine. N-demethylase were significantly lowered during pregnancy. In contrast with the greatly impaired induction of delta-aminolaevulinate synthase, the induction of haem oxygenase in response to CoCl2 remained unaltered in pregnant rats. The normal perturbations of delta-aminolaevulinate synthase, consisting of an initial inhibition followed by a rebound increase in the enzyme activity associated with CoCL2 treatment, were observed during pregnancy. These findings indicate that hormones and metabolic factors associated with gestation exert significant but differential controls on the induction patterns of delta-aminolaevulinate synthase and haem oxygenase.