Clinicoepidemiological,Toxicological, and Safety Evaluation Studies on Argemone Oil

Abstract

Consumption of oil extracted from accidental or deliberate contamination of argemone seed to mustard seed is known to pose a clinical condition popularly referred to as Epidemic Dropsy. Several outbreaks of Epidemic Dropsy have occurred in the past in India as well as in Mauritius, Fiji Island, and South Africa. Clinico-epidemiological manifestations of argemone oil poisoning include vomiting, diarrhea, nausea, swelling of limbs, erythema, pitting edema, breathlessness, etc. In extreme cases, glaucoma and even death due to cardiac arrest have been encountered. The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Histopathological studies suggest that liver, lungs, kidney, and heart are the target sites for argemone oil intoxication. Studies have shown to elucidate the cocarcinogenic potential of argemone oil that can be correlated with the binding of sanguinarine with a DNA template. Pharmacological response in intestine revealed immediate stimulation of tone and peristaltic movements of the gut in the sanguinarine-treated animals. Argemone oil/Sanguinarine caused a decrease in hepatic glycogen levels which may be due to the activation of glycogenosis leading to an accumulation of pyruvate in the blood of Epidemic Dropsy cases. The increase in pyruvate levels causes uncoupling of oxidative phosphorylation leading to breathlessness, as observed in patients. Sanguinarine has been shown to inhibit Na⁺, K⁺-ATPase activity of different organs such as brain, heart, liver, intestine, and skeletal muscle, which may be due to the interaction with the glycoside receptor site on ATPase enzyme, thereby causing a decrease in the active transport of glucose. Argemone oil/alkaloid showed a Type II binding spectra with hepatic cytochrome P-450 (P-450) protein, thereby causing loss of P-450 content and an impairment of phase I and phase II enzymes. A green fluorescent metabolite of sanguinarine, benzacridine was detected in the milk of grazing animals. The delayed appearance of this metabolite in urine and feces of experimental animals suggests the slow elimination of the alkaloid. Argemone oil enhances hepatic microsomal and mitochondrial lipid peroxidation, indicating that these two organelles are the sites of membrane damage. Furthermore, studies suggest that singlet oxygen and hydroxyl radical are involved in argemone oil toxicity. Several bioantioxidants show protective effect in argemone oil-induced toxicity in experimental animals. The line of treatment in argemone-intoxicated epidemics has so far been only symptomatic, and specific therapeutic measures are still lacking, although it has been suggested that diuretics, bioantioxidants, steroids, vitamins, calcium- and protein-rich diet had some beneficial effects on Epidemic Dropsy cases.     

Inhibitory effects of nilutamide, a new androgen receptor antagonist, on mouse and human liver cytochrome P-450

The effects of nilutamide were studied first with human liver microsomes. At concentrations expected in the human liver (110 microM), nilutamide inhibited hexobarbital hydroxylase, benzphetamine N-demethylase, benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities by 85, 40, 35 and 25%, respectively. There was no in vitro inhibition of NADPH-cytochrome c reductase activity, no in vitro loss of CO-binding cytochrome P-450, and no spectral evidence for the in vitro formation of a possible cytochrome P-450Fe(II)-nitroso metabolite complex. Other studies were performed with mouse liver microsomes. Nilutamide (550 microM) did not significantly increase the consumption of NADPH by aerobic microsomes, and did not modify the kinetics for the reduction of cytochrome P-450 by NADPH-cytochrome P-450 reductase in an anaerobic system. Nilutamide (22 microM) produced either a type I or a type II binding spectrum. Kinetics for the inhibition of hexobarbital hydroxylase were consistent with competitive inhibition. A last series of experiments was performed after administration of nilutamide in mice. Thirty minutes after administration of doses (15 or 30 mumol.kg-1 i.p.) similar to those used in humans, the hexobarbital sleeping time was increased by 40 and 60%, respectively. There was no evidence, however, for the irreversible inactivation of microsomal enzymes since CO-binding cytochrome P-450 and monooxygenase activities remained unchanged in liver microsomes from mice killed 1 or 6 hr after administration of nilutamide (30 mumol.kg-1 i.p.). These results show that nilutamide inhibits hepatic cytochrome P-450 activity, and suggest that inhibition may actually occur after therapeutic doses of nilutamide in humans.     

Dietary fat--a requirement for induction of mixed-function oxidase activities in starved-refed rats

Male rats were starved 0-48 hr, and then refed diets containing 0% (F.F.) to 20% corn oil (C.O.) lab chow or 20% coconut oil (C.C.O.) for 1-4 days. Some received phenobarbital sodium (80 mg/kg, i.p. daily) for 1-3 days prior to decapitation. Five cytochrome P-450-dependent indicators were assayed as measures of altered hepatic microsomal function: ethylmorphine N-demethylase (EMDM), N-nitrosodimethylamine (DMN)-N demethylase, aniline hydroxylase (AH), benzo[a]pyrene hydroxylase (AHH) and CO-difference spectra (P-450). Increasing dietary corn oil (0, 0.5, 10, 20%) in control rats resulted in a progressive increase in the activities of these five enzymes. Dietary fat influenced phenobarbital (Pb) inducibility of all mixed-function oxidase (MFO) enzymes measured except AHH. Pb induced the remaining enzymes only 11-22% in animals fed fat-free diet as compared to 119-246% in animals fed coconut oil and corn oil. Rats fed fat-free diet for 21 days without prior food deprivation and administered Pb had 79% more EMDM, 34% more AH and 120% more P-450 than non-induced controls, whereas rats fed 20% corn oil diet had 227% more EMDM, 143% more AH and 128% more P-450. A requirement of dietary fat for induction of MFO by Pb was demonstrated by these starvation-refeeding experiments. Coupled with data recovered from the 21-day studies, these experiments suggest that a compensatory mechanism may be operative during chronic feeding of the fat-free diet to partially return inducibility to the drug-metabolizing system.     

Imipramine prevention of carbon tetrachloride-induced liver necrosis at late states of the intoxication process

Imipramine administration (50 mg kg-1, i.p.) to Sprague-Dawley male rats (240-290 g) 6 or 10 h after CCl4 (1 ml kg-1, i.p.) partially prevents liver necrosis induced by the hepatotoxin. When imipramine is given 30 min before CCl4, it inhibits in part the CCl4-induced lipid peroxidation and the covalent interactions of reactive metabolites with microsomal lipids or proteins and partially prevents CCl4-induced cytochrome P-450 destruction, but not glucose 6 phosphatase activity depression. Imipramine administration prior to CCl4 does not modify levels of the hepatotoxin reaching the liver or the body temperature of CCl4 treated animals. Early preventive effects of imipramine on cytochrome P-450, might be attributed to inhibition of covalent interactions of reactive metabolites. The hypothesis that imipramine exerted late preventive effects by interfering with calcium deleterious effects or by modulation of protein and phospholipid synthesis or degradation is analyzed.     

Alterations in xenobiotic metabolizing enzymes in brain and liver of rats coexposed to endosulfan and malathion

The effects of endosulfan (3 mg kg-1 body wt., i.p.) and malathion (30 mg kg-1 body wt.) and their coexposure on rat hepatic and brain xenobiotic metabolizing enzymes were investigated. Endosulfan was found to induce aminopyrine-n-demethylase (81%) and aniline hydroxylase (59%) activities significantly in liver and to a lesser extent in brain. Malathion treatment induced malathion carboxylesterase activity in both liver (50%) and brain (22%), significantly depleted liver glutathione (35%) content with stimulation of glutathione-S-transferase (50%) and inhibited the activity of mixed-function oxidases. In the coexposed animals, malathion's inhibitory influence on mixed-function oxidases and endosulfan's inhibitory effect on malathion carboxylesterase were found to dominate, while endosulfan potentiated the activity of glutathione-S-transferase significantly in liver (69%) and brain. A similar trend of alteration in coexposed brain was found, but to a lesser extent. A significant inhibition in brain acetylcholine esterase activity (42%) in the coexposed animals suggests that endosulfan may potentiate the toxicity of malathion by interfering with glutathione and carboxylesterase routes of malathion detoxification.     

Regulation of renal cytochrome P-450. Effects of two-thirds hepatectomy, cholestasis, biliary cirrhosis and post-necrotic cirrhosis on hepatic and renal microsomal enzymes

The possibility of a relationship between hepatic and renal cytochrome P-450 contents was assessed in rats with liver disease. In rats killed 3 days after two-thirds hepatectomy (a model for hepatocellular insufficiency), the total microsomal cytochrome P-450 content of the whole liver was decreased by 60% as compared to that in control rats; renal cytochrome P-450 was increased by 30% while the 7-ethoxycoumarin deethylase activity of kidney microsomes was increased by 80%. In rats killed 7 days after bile duct ligation (a model for cholestasis) or 35 days after bile duct ligation (a model for biliary cirrhosis), hepatic cytochrome P-450 was decreased by 60% and 45%, respectively, while renal cytochrome P-450 content was increased by 50% and 150%, respectively. In contrast, in rats killed 15 days after the last dose of carbon tetrachloride, 1.3 ml/kg twice weekly for 3 months (a model for post-necrotic cirrhosis), both hepatic and renal cytochrome P-450 contents remained unchanged. Phenobarbital (80 mg/kg daily for 3 days) was a poor inducer of renal cytochrome P-450 in sham-operated rats but became a potent inducer of renal cytochrome P-450 in rats with two-thirds hepatectomy. We conclude that renal cytochrome P-450 is increased in three models in which hepatic cytochrome P-450 contents are decreased (two-thirds hepatectomy, cholestasis and biliary cirrhosis), but remains unchanged in a model of severe liver pathology, in which hepatic cytochrome P-450 content is not modified (late, post-necrotic cirrhosis). The hypothetical role of endogenous inducer(s) is discussed.     

Depression of mouse liver microsomal mixed function oxidase enzymes by adriamycin

Levels of cytochrome P-450 and cytochrome b5, and activities of NADPH cytochrome c reductase and 7-ethoxycoumarin O-deethylase, were found to be significantly decreased in hepatic microsomes prepared from mice killed 24 h after administration of a single intraperitoneal (i.p.) dose of adriamycin (ADR, 5 mg/kg). In contrast, both ascorbate-induced lipid peroxidation and conjugated dienes were increased in the same preparations. In vitro addition of ADR (5 micrograms/ml) to hepatic microsomal preparations (1 mg/ml protein) from the control mice also led to a substantial decrease in the mixed function oxidase (MFO) enzymes. A characteristic spectral change with an absorption peak at 408 nm and trough at 422 nm was associated with this in vitro interaction. It is suggested that the loss of cytochrome P-450 and related MFO enzymes due to ADR treatment is related to the generation of free radicals and subsequent lipid peroxidation in the liver.     

Disulfiram prevents acetaminophen hepatotoxicity in rats

Hepatic necrosis due to an oral acetaminophen overdose (4.25 g/kg b.wt.) was prevented by pretreatment with disulfiram 100 mg/kg, given for 3 weeks or as a single dose. Twenty-four hours after acetaminophen the impairment of hepatic function, measured as prothrombin index, and the depletion of hepatic glutathione were prevented. Hepatic cytochrome P-450 levels were unchanged but cytochrome P-450 mediated p-nitroanisole demethylation was reduced by disulfiram pretreatment. Disulfiram pretreatment reduced 24 hour urinary excretion of acetaminophen-mercapturate and- cysteine while excretion of -sulfate and -glucuronide was unchanged. After 72 hours acetaminophen induced hepatic necrosis were prevented. Identical observations were made in animals pretreated with disulfiram for 3 weeks. Five hours after acetaminophen overdose its irreversible binding to hepatic proteins was not changed. After 24 hours, however, it was increased in animals pretreated with a single disulfiram dose and unchanged in animals pretreated for 3 weeks. The protective mechanism of disulfiram after acetaminophen overdose is not mediated via a change in overall irreversible binding of acetaminophen to hepatic protein.     

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats was examined with respect to the mixed function oxidase system, anaerobic dehalogenation activity of halothane, and the antioxidant system. The levels of cytochrome P-450 and NADPH-cytochrome P-450 reductase were significantly higher in guinea pigs than in rats. There was no difference between the two animals in anaerobic dehalogenation activity of halothane per cytochrome P-450 in microsomes. Microsomal alpha-tocopherol was significantly lower in guinea pigs than in rats, and was increased by multiple exposure to halothane in guinea pigs but remained lower than in rats. Microsomal alpha-tocopherol was decreased in rats by multiple exposure. The concentration of reduced glutathione and ascorbic acid was decreased significantly by multiple exposure to halothane in guinea pigs but not in rats. These results suggest that the higher level of halothane-induced hepatic microsomal lipid peroxidation in guinea pigs is due to the large production of radical metabolites resulting from the large amounts of cytochrome P-450, the high activity of NADPH-cytochrome P-450 reductase, and the low concentration of microsomal alpha-tocopherol.     

Depression of the hepatic cytochrome P-450 monooxygenase system by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The effects of the neurotoxic compound, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the hepatic cytochrome P-450 monooxygenase system were assessed using C57 BL/6J mice. Treatment with MPTP caused a marked depression of hepatic cytochrome P-450 content, ethoxyresorufin O-dealkylase and NADPH cytochrome C reductase activities. This effect was maximal 3 to 6 hours after treatment and was dependent on the dose of MPTP administered. Depression of spectrophotometrically measured cytochrome P-450 content was associated with increase in cytochrome P-420 content and lipid peroxidation. In vitro studies showed the formation of a metabolic-intermediate complex with cytochrome P-450 which may partially explain the depression of cytochrome P-450 content and activity by MPTP.     

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.     

Response of hepatic microsomal mixed-function oxidases to various types of insecticide chemical synergists administered to mice

For several hours after a single intraperitoneal (i.p.) dose to mice, piperonyl butoxide, 2-methylpropyl 2-propynyl phenylphosphonate (NIA 16824), and 5,6-dichloro-l, 2,3-benzothiadiazole (WL 19255) inhibit hepatic microsomal mixed-function oxidase (mfo) activity and the apparent level of cytochrome P-450 but, after 24–72 hr, they induce increased mfo activity and P-450 content. Other types of insecticide chemical synergists studied are generally less active or inactive in this respect. The active compounds enhance the toxicity of several insecticide chemicals and markedly prolong hexobarbital sleeping time but not hexobarbital toxicity. The action of WL 19255 differs from that of piperonyl butoxide and NIA 16824 in that it is relatively non-specific in inhibiting enzymatic activity on several substrates, gives a more prolonged effect on P-450, and the magnitude of induction is greater.     

Biochemical toxicology of argemone alkaloids. III. Effect on lipid peroxidation in different subcellular fractions of the liver

Consumption of edible oils contaminated with Argemone mexicana seed oil causes various toxic manifestations. In this investigation the in vivo effect of argemone oil on NADPH-dependent enzymatic and Fe2+-, Fe2+/ADP- or ascorbic acid-dependent non-enzymatic hepato-subcellular lipid peroxidation was studied. Parenteral administration of argemone oil (5 ml/kg body weight) daily for 3 days produced a significant increase in both non-enzymatic and NADPH-supported enzymatic lipid peroxidation in whole homogenate, mitochondria, and microsomes. Lipid peroxidation aided by various pro-oxidants, namely Fe2+, Fe2+/ADP and ascorbic acid also revealed a significant enhancement in the whole homogenate, mitochondria and microsomes of argemone oil-treated rats. Further, when compared with whole homogenate, the hepatic mitochondria and microsomes of either control or argemone oil-treated rats showed a 4- and 6-fold increase in non-enzymatic, and a 5- and 18-fold increase in NADPH-dependent enzymatic lipid peroxidation, respectively. Similarly, both mitochondrial and microsomal fractions showed a 5- and 7-fold increase in Fe2+-, and a 12- and 15-fold increase in either Fe2+/ADP- or ascorbic acid-aided lipid peroxidation, respectively. These results suggest that the hepatic microsomal as well as the mitochondrial membrane is vulnerable to the peroxidative attack of argemone oil and may be instrumental in leading to the hepatotoxicity symptoms noted in argemone poisoning victims.     

Protective effects of selenium on acetaminophen-induced hepatotoxicity in the rat

Experiments were undertaken to examine the ability of selenium to protect against acetaminophen-induced hepatotoxicity and to examine possible mechanisms for this protective effect. Pretreatment of male, Sprague-Dawley rats with sodium selenite (12.5 mumol Se/kg, ip) 24 hr prior to acetaminophen administration produced a significant protection against the hepatotoxic effects of acetaminophen as assessed by a decrease in the plasma appearance of alanine aminotransferase and aspartate aminotransferase activities following acetaminophen. This was accompanied by an increase in the hepatic glutathione levels in selenium-treated animals and an inhibition in the decrease in hepatic glutathione content observed in animals receiving hepatotoxic doses of acetaminophen. Selenium pretreatment decreased the in vivo covalent binding of acetaminophen metabolites to hepatic protein, but did not alter hepatic microsomal cytochrome P-450 content or NADPH cytochrome c reductase activity, suggesting that selenium does not significantly alter the metabolism of acetaminophen to reactive electrophilic metabolites by the cytochrome P-450-dependent mixed-function oxidase enzyme system. Selenium produced an increase in the activity of gamma-glutamylcysteine synthetase which may account for the increased glutathione availability in selenium-treated animals and increased the activities of glutathione S-transferase and glucose-6-phosphate dehydrogenase. Examination of the urinary metabolite profile in selenium-treated animals revealed that the urinary excretion of acetaminophen and its metabolites was significantly increased over a 72-hr period. The increase occurred in the AAP-glucuronide metabolite while parent AAP and AAP-sulfate were actually decreased in selenium-treated rats. No change in recovery was observed in the AAP-glutathione or AAP-mercapturate urinary metabolites. While the glutathione conjugating system is enhanced by selenium treatment, amelioration of acetaminophen toxicity is most likely the result of enhanced glucuronidation which effectively diverts the amount of acetaminophen to be converted by the cytochrome P-450 system to the toxic metabolite.     

Depressant effects of the interferon inducer polyriboinosinic:polyribocytidylic acid on cytochrome P-450 hemoproteins

Partially purified fractions of cytochrome P-450 were prepared from hepatic microsomes recovered from male rats 12 h after administration of either saline or polyriboinosinic:polyribocytidylic acid (poly I:C). Poly I:C reduced the microsomal concentration of cytochrome P-450 by 19% and decreased the maximal binding spectrum (delta Amax) resulting from addition of the type-II substrate 2,4-dichloro-6-phenylphenoxyethylamine to one fraction (B2) while increasing the affinity of that fraction for this substrate. Poly I:C also reduced the microsomal hydroxylation of benzo(a)pyrene and the N-demethylation of benzphetamine by the other fraction (B1). Since 14C-leucine incorporation into cytochrome P-450 was increased in poly I:C-treated rats, it is suggested that poly I:C depresses hepatic mixed-function oxidase activity by increasing the rate of degradation of specific cytochrome P-450s.     

Effects of the immunosuppressant FK-506 and its analog FK-520 on hepatic and renal cytochrome P450 mixed-function oxidase.

The novel immunosuppressant FK-506 and its analog FK-520 were found to inhibit the hepatic microsomal mixed-function oxidase system in male Sprague-Dawley rats. At 5 and 10 mg/kg/day, s.c., for 6 days they caused 30-80% decreases in cytochrome P450 levels, NADPH-cytochrome P450 reductase, and benzphetamine N-demethylase activities. The metabolism of FK-506 itself was inhibited by 50%. FK-506 and FK-520 had a minimal effect on the renal cytochrome P450 levels unlike cyclosporin A which produced a 67% increase after six daily 25 mg/kg doses. A single dose of FK-506 (25 mg/kg, s.c.) had a minimal effect on the hepatic or renal metabolizing enzyme system. In vitro, addition of FK-506 and FK-520 to human and control rat liver microsomes resulted in a concentration-dependent inhibition of benzphetamine N-demethylation (10-20% at 50 microM, 60-75% at 250 microM). We suggest that in view of its potential to inhibit hepatic cytochrome P450-dependent mixed-function oxidase, resulting in the inhibition of its own metabolism, FK-506 should be administered with caution to transplant patients.     

THe formation of cytochrome P-450-metabolic intermediate complexes from amines, in the isolated perfused rat liver.

1. Cytochrome P-450-metabolic intermediate (MI) complexes were formed from SKF 525-A, propoxyphene, acetylmethadol, noracetylmethadol, norbenzphetamine, and N-hydroxyamphetamine, but not methadone, in the isolated perfused rat liver. 2. The amount of MI complex from SKF 525-A (8% of the cytochrome P-450) after 1 h exceeded that for all other compounds (1--2%). 3. Both MI complex and residual substrate contributed to the inhibition of mixed-function oxidase activity observed. No substrate altered the total cytochrome P-450 concentration of NADPH-cytochrome c reductase activity. 4. The formation of MI complexes in isolated perfused liver corresponds to that seen in whole animals, and contrasts with that seen in microsomal preparations.     

21-aminosteroids prevent the down-regulation of hepatic cytochrome P450 induced by hypoxia and inflammation in conscious rabbits.

1 This study was conducted to assess whether a 21-aminosteroid, U74389G, could prevent the down-regulation of hepatic cytochrome P450 (P450) induced by acute moderate hypoxia or an inflammatory reaction. 2 The rabbits of two groups (n = 6 per group) were subjected to acute moderate hypoxia (PaO2 approximately 35 mmHg), one pre-treated with U74389G (3 mg kg-1 i.v. every 6 h, for 48 h). The rabbits of two other groups received 5 ml of turpentine s.c., one of them being pre-treated with U74389G (3 mg kg-1 i.v. every 6 h, for 72 h). The kinetics of theophylline (2.5 mg kg-1) were assessed to evaluate the activity of the P450. Once the rabbits were sacrificed, the P450 content and the amount of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were estimated in the liver. 3 Compared with control rabbits, hypoxia and inflammation increased theophylline plasma concentrations, as a result of a decrease in theophylline systemic clearance (P<0.05). Both experimental conditions reduced hepatic content of P450 by 40-50% (P<0.05) and increased the amount of hepatic TBARS by around 50% (P<0.05). Pre-treatment with U74389G prevented the hypoxia- and inflammation-induced decrease in theophylline systemic clearance, the down-regulation of hepatic P450, and the increase in liver TBARS. 4 It is concluded that in the rabbit, U74389G prevents hepatic P450 depression produced by acute moderate hypoxia and a turpentine-induced inflammatory reaction, possibly by eliciting a radical quenching antioxidant activity.     

The Inhibition and Potentiation of Procarbazine on Hepatic Mixed-Function Oxidases in Pentobarbital Tolerant and Nontolerant Mice

ABSTRACT

The effect of procarbazine on mixed-function oxidases was investigated in naive and pentobarbital tolerant mice. In mice receiving procarbazine, 200 mg/kg, i.p. 1 hr earlier, metabolisms of pentobarbital, aniline and ethylmorphine in vitro and cytochrome P-450 content of hepatic microsomes were significantly decreased. The drug binding of either aniline or pentobarbital to cytochrome P-450 was also decreased. However, procarbazine failed to exert this effect after the enzymes had been induced by continuous administration of pentobarbital. Interestingly, procarbazine enhanced the barbiturate induced hepatic microsomal mixed-function oxidase activities when it was administered before the implantation of pentobarbital pellet. Both cytochrome P-450 and cytochrome b₅ content after pentobarbital pellet implantation were further increased by pretreatment with procarbazine. This finding was further substantiated by the increase in pentobarbital and aniline binding to cytochrome P-450. The present studies may provide another model for studying the nature of hepatic mixed-function oxidase induction process.     

Degradation of cytochrome P-450 heme in ascorbic acid-deficient guinea pigs.

Degradation of hepatic cytochromc P-450 heme in ascorbic acid-deficient and ascorbic acid-dosed (p.o., 25 mg/100 g/body wt/day) guinea pigs was investigated by determining the turnover of radioactive cytochrome P-450 and the formation of in vivo expired carbon monoxide (¹⁴CO). ¹⁴CO is a specific degradation product of the labeled heme. Hepatic levels of cytochrome P-450 and cytochrome b₅ were decreased significantly (61 per cent, P < 0.001. and 29 per cent. P < 0.01, respectively) in scorbutic animals compared to ascorbic acid-adequate animals. After the administration of δ-amino-levulinic acid [3, 5-³H], the disappearance of radioactivity from the fast-phase and slow-phase components of cytochrome P-450 heme (CO-binding pigments) exhibited half-lives of 4.2 and 30.8 hr, respectively, in the ascorbic acid-depleted guinea pigs. The turnover of cytochrome P-450 heme was similar for both phases in animals fed the ascorbic acid-deficient diet and dosed with ascorbic acid or animals fed a normal stock diet. In guinea pigs deficient in ascorbic acid and injected with [5-¹⁴C]δ-aminolevulinic acid, the cumulative expired ¹⁴CO was similar to guinea pigs adequate in ascorbic acid. These studies demonstrate that the decrease in hepatic cytochrome P-450 content in ascorbic acid deficiency is not due to a defect in cytochrome heme catabolism. The role of ascorbic acid in stabilizing cytochrome P-450 remains to be determined.