Addition Of SKF-525A Research Articles

Ciprofloxacin-induces free radical production in rat cerebral microsomes

In the presence of ciprofloxacin (CPFX), free radical adduct formation was demonstrated in rat cerebral microsomes using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone by electron spin resonance spectroscopy. Active microsomes, dihydronicotinamide-adenine dinucleotide phosphate, and ciprofloxacin were necessary for the formation of a spin trap/radical adduct. Adduct formation increased dose-dependently at 0.5–1 mM CPFX concentration for 180 min, and 0.3–1 mM concentration level for 240 min. The addition of SKF 525A, ZnCl2 or desferrioxamine to the incubation system caused complete inhibition of the radical formation. However, pretreatment of microsomal system with superoxide dismutase (SOD) did not induce any protective effect. Induction of lipid peroxidation, and depletion of thiol levels by CPFX were also shown in the system. These results strongly suggested that CPFX produces free radical(s) in the cerebral microsomes of rats.

Celecoxib enhances the detoxification of diethylnitrosamine in rat liver cancer

To study the effect of celecoxib (CXB) on diethylnitrosamine activation through the regulation of cytochrome P450 in a hepatocarcinogenesis model. Six-week-old male Sprague-Dawley rats were randomly divided into five groups, a non-treated group (NT), a diethylnitrosamine-treated group (DEN), a DEN+CXB-treated group (DEN+CXB), and CXB 8 d-treated and CXB 32 d-treated groups. The effects of celecoxib on the enzymatic activities of CYP1A1, 2A, 2B1/2, and 2E1 were assessed in hepatic microsomes 24 h after DEN administration. Changes in CYP1A1 and CYP2B1/2 protein expression were also evaluated. The rate of DEN metabolism was measured by the production of the deethylation metabolite acetaldehyde, and the denitrosation metabolite nitrite. DEN+CXB administration produced a significant increase in the enzymatic activities of CYP2B1/2 and 1A1, whereas it did not change the activities of CYP2A and 2E1, compared to that of the DEN group. CXB treatment for eight days did not produce a significant effect on enzymatic activity when compared to the NT group; however, when it was administered for prolonged times (CXB 32 d group), the enzymatic activities were increased in a similar pattern to those in the DEN+CXB group. The observed increase in the enzymatic activities in the DEN+CXB group was accompanied by an increase in the CYP2B1/2 protein levels; no changes were observed in the levels of CYP1A1. In vitro, CXB increased the denitrosation of DEN, a pathway of metabolic detoxification. The addition of SKF-525A, a preferential inhibitor of CYP2B, abrogated the denitrosation of DEN. These results suggest that the mechanism of action of CXB involves enhancement of the detoxification of DEN by an increasing denitrosation via CYP2B1/2.

Metabolites of Hirsuteine and Hirsutine, the Major Indole Alkaloids of Uncaria rhynchophylla, in Rats

The metabolic fate of hirsuteine (HT) and hirsutine (HS), the major indole alkaloids of Uncaria rhynchophylla, was investigated using rats. On HPLC analysis, urine from rats orally administered HT were found to contain two metabolites (HT1 and HT2) together with unchanged HT. Similarly HS also was metabolized to two compounds (HS1 and HS2). Metabolite structures were determined to be 11-hydroxyhirsuteine-11-O-beta-D-glucuronide (HT1), 11-hydroxyhirsuteine (HT2), 11-hydroxyhirsutine-11-O-beta-D-glucuronide (HS1) and 11-hydroxyhirsutine (HS2), based on spectroscopic and chemical data. HT1 and HS1 were also detected in bile from rats administered HT and HS, respectively. Total cumulative urinary excretion within 72 h of oral administration was approximately 14% and 26% of the HT and HS doses, respectively, while total cumulative biliary excretion was 35% and 46%, respectively. HT and HS 11-hydroxylation were catalyzed by rat liver microsomes. This 11-hydroxylation activity was inhibited by addition of SKF-525A (a nonselective CYP inhibitor) or cimetidine (a CYP2C inhibitor). These results indicate that orally administered HT and HS are converted to 11-hydroxy metabolites in rats, and that the metabolites are predominantly excreted in bile rather than urine following glucuronidation. Furthermore, the results suggest that CYP2C enzymes are involved, at least in part, in the specific 11-hydroxylation of HT and HS.

Chlorpropham induces mitochondrial dysfunction in rat hepatocytes.

The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) and its metabolites were studied in freshly isolated rat hepatocytes and isolated rat hepatic mitochondria, respectively. The exposure of hepatocytes to CIPC caused a concentration (0.25-1.0 mM)- and time (0-3h)-dependent cell death accompanied by loss of cellular ATP and adenine nucleotides. CIPC at a weakly toxic level (0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to a minor metabolite 3-chloroaniline (3CA). The addition of SKF-525A (50 microM), an inhibitor of microsomal monooxygenase, enhanced the CIPC (0.5 mM)-induced cytotoxicity accompanied by loss of ATP and 4OH-CIPC and inhibited the decrease in the concentration of the parent compound. CIPC led to a strong decrease in cellular ATP content compared to its metabolites, 4OH-CIPC and 3CA. On the other hand, the exposure of isolated hepatic mitochondria to CIPC reduced State 3 respiration with a FAD-linked substrate (succinate plus rotenone) and/or with a NAD+ -linked substrate (pyruvate plus malate), whereas State 3 respiration with ascorbate plus tetramethyl-p-phenylendiamine (cytochrome oxidase-linked respiration) was not affected markedly by CIPC. Further, the addition of CIPC caused an increase in the rate of State 4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner, respectively. In contrast, the addition of neither 4OH-CIPC nor 3CA markedly affected the rate of states 3 and/or 4 oxygen consumption. These results indicate that CIPC-induced cytotoxicity is mediated by the parent compound rather than by its metabolites such as 4OH-CIPC and 3CA, and that the toxicity is associated with a rapid depletion of ATP via impairment of mitochondrial function related to oxidative phosphorylation.

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 °C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N, N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.

RENAL METABOLISM OF ACRYLONITRILE TO CYANIDE: IN VITRO STUDIES

Acrylonitrile (VCN) is a widely used industrial chemical. The present work examines the mechanism of its renal toxicity. In renal centrifugal fractions from Sprague–Dawley rats, the metabolism of VCN to cyanide (CN−) was highest in the microsomal fraction and required a NADPH-generating system in the presence of magnesium ions for maximum activity. This biotransformation of VCN to CN−was characterised with respect to time (15 min), microsomal protein concentration (3 mg ml−1), pH (7.5) and temperature (37°C). The Vmaxof the reaction was 118.2 pmol CN−mg−1protein min−1and Kmwas 160.2 μmol VCN. Activation of VCN to CN−was markedly increased in microsomes obtained from phenobarbital (PB), ethanol, 4-methylpyrazole and 3-methylcholanthrene-treated rats by 161.5, 89.6, 71.0 and 50.2%, respectively. Addition of SKF 525-A (5×10−4m) or benzimidazole (2 m m) to the incubation mixtures significantly inhibited VCN metabolism by 66.6 and 78.8%, respectively. VCN metabolism to CN−was enhanced significantly by the addition of 10 m m of glutathione (GSH), l -cysteine, d -penicillamine, cysteamine or 2-mercaptoethanol to 389.5, 886.5, 611.1, 145.5 and 384.0% of control, respectively. These findings indicate that VCN is metabolised in the kidney via cytochrome P-450-dependent mixed function oxidase system. 1999 Academic Press@p$hr

Presystemic intestinal metabolism of N-nitrosodimethylamine in mouse intestine.

N-Nitrosodimethylamine (NDMA), a common food contaminant, is a potent liver carcinogen in rodents. A high presystemic intestinal metabolism has been shown for several nitrosamines including environmentally important compounds. We determined the metabolism of 1 micron [14C]-NDMA in isolated perfused mouse intestinal segments. We found NDMA to be equally distributed between the absorbed fluid and the perfusate. During a 2-h perfusion period, 0.13% of the radioactivity was converted to CO2. The formation of CO2 was decreased by pretreatment with diallylsulfide or addition of SKF 525A, and slightly increased by phenobarbital. Hydrophilic metabolites were found in the absorbate (0.9%) and perfusate (3.8%) of untreated mice. The amount of metabolites in the absorbate was increased by treatment with acetone or phenobarbital (8-fold), but not after starvation, with formaldehyde being present only in phenobarbital-treated animals. Treatment with diallylsulfide or addition of SKF 525A reduced the amount of metabolites in acetone-treated animals to control values. In conclusion, intestinal turnover does not significantly reduce the body burden of orally ingested NDMA and thus is not a first-line defense against this carcinogenic nitrosamine. NDMA metabolism has been attributed to the presence of cytochrome P450IIE1, which has not been detected in the intestine of untreated animals. The low turnover of NDMA, the induction by acetone and phenobarbital treatment, and the inhibition by diallylsulfide suggest the presence of low amounts of this or related cytochrome P450 isozyme(s) in mouse intestine.

IN-VITROTESTICULAR BIOACTIVATION OF ACRYLONITRILE

The present work examines the mechanism of testicular toxicity of acrylonitrile. In testicular centrifugal fractions from Sprague Dawley rats, the metabolism of VCN to cyanide (CN−) was highest in the microsomal fraction and required NADPH for maximum activity. This biotransformation of VCN to CN−was characterized with respect to time (30min), microsomal protein concentration (1.5mgml−1), pH (7.5) and temperature (37°C). The Vmaxof the reaction was 65.1pmol CN−mg protein−1min−1and Kmwas 88.6gmmol VCN. Flushing the microsomes with carbon monoxide (CO)(4:1, CO/O2v/v), addition of benzimidazole (1mM) or addition of SKF 525-A (5×10−4M) to incubation mixtures significantly inhibited VCN metabolism by 49%, 54% and 37.4% respectively. Activation of VCN to CN−was markedly increased in microsomes obtained from phenobarbital (PB)-treated rats (128.2%). Addition of glutathione (GSH),L-cysteine,D-penicillamine or 2-mercaptoethanol significantly enhanced the release of CN−from VCN 126%, 247%, 202% and 129% of the control value respectively. These findings indicate that VCN is metabolized in the testis via cytochrome P-450 dependent mixed function oxidase system.

Lipid Peroxidation and Cherniluminescence during Naproxen Metabolism in Rat Liver Microsomes

1. Rat liver microsomal suspension containing NADPH and MgCl2 was incubated at 37 degrees C with naproxen, a non-steroidal anti-inflammatory drug. Thiobarbituric acid reactive substances (TBA-RS), high molecular weight protein aggregates and fluorescent substances were formed in the microsomal suspension. 2. Chemiluminescence was produced from the microsomal suspension. This chemiluminescence production was well correlated to the TBA-RS formation, indicating that the chemiluminescence production was closely associated with the lipid peroxidation. 3. The addition of SKF-525A to the microsomal suspension inhibited the production of TBA-RS, chemiluminescence and 6-demethylnaproxen (6-DMN), the oxidative product of naproxen. Further, the antioxidant, alpha-tocopherol and singlet oxygen quenchers like histidine, dimethylfuran and 1,4-diazabicyclo[2,2,2]octane strikingly inhibited the productions of chemiluminescence and TBA-RS. 4. Neither naproxen nor 6-DMN caused lipid peroxidation in the absence of NADPH. Thus, lipid peroxidation and chemiluminescence during the oxidation of naproxen in liver microsomes was suggested to be provoked by reactive oxygen species and an origin of chemiluminescence was shown to be singlet oxygen.

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.

Metabolic activation of lidocaine and covalent binding to rat liver microsomal protein

Incubation of [ 14C]lidocaine with rat liver microsomes in the presence of an NADPH-generating System resulted in covalent bindings of a 14C-labelled material to microsomal protein. The covalent binding of radioactivity needed NADPH and atmospheric oxygen, and was diminished by purging of carbon monoxide and the addition of SKF-525A. Hence the covalent binding of a 14C-labelled material resulting from a reactive metabolite of lidocaine formed by cytochrome P450-dependent monooxygenation. The covalent binding measured at various concentrations of lidocaine (2.5–30 μM) followed Michaelis-Menten kinetics, and the K m , value (4.52 μM) of the activation reaction was close to the K m value (1.78μM) of lidocaine 3-hydroxylation. the metabolism-dependent covalent binding of lidocaine to microsomal protein as well as lidocaine 3-hydroxylase activity was much lower in the Dark Agouti strain rat, which is known as a poor-metabolizer animal model of debrisoquine 4-hydroxylation, than in the Wistar rat for the corresponding sexes. the covalent binding in male rats was greater than that in females of both strains, but the extent of the sex difference in the binding was smaller than that of the lidocaine N-deethylase activity in Wistar rats. Propranolol and quinidine, specific inhibitors of debrisoquine 4-hydroxylase, markedly inhibited lidocaine 3-hydroxylase activity of Wistar male rats, but not N-deethylase activity. These compounds also inhibited the metabolism-dependent covalent binding of lidocaine to microsomal protein. These strain difference and inhibition studies showed that the reaction converting lidocaine to a reactive metabolite capable of binding covalently to microsomal protein was related to lidocaine 3-hydroxylation, and may be catalysed by cytochrome P450 isozyme(s) belonging to the CYP2D subfamily. The covalent binding of radioactivity to rat liver microsomal protein was diminished by nucleophiles, reduced glutathione and cysteine, indicating that the reactive metabolic intermediate of lidocaine is an electrophilic metabolite such as an arene oxide.

In vitro studies on the metabolism and co valent binding of [ 14C]1,1-dichloroethylene by mouse liver, kidney and lung

The metabolism and covalent binding of 1,1-dichloro[1,2- 14C]ethylene (DCE) to subcellular fractions of liver, kidney and lung of C57BL/6N mice have been investigated in vitro. Covalent binding was NADPH- and cytochrome P-450-dependent. The microsomal fraction bound more radiolabel than any other subcellular fraction, and the levels of covalent binding in cell fractions correlated well with their cytochrome P-450 content. Covalent binding by mouse liver and lung microsomes also reflected their cytochrome P-450 content. However, although mouse kidney microsomes contained twice as much total cytochrome P-450 as the lung, no detectable covalent binding of DCE-derived radioactivity occurred in kidney. Omission of NADPH, heat inactivation of microsomes, carbon monoxide, addition of SKF-525A, piperonyl butoxide or reduced glutathione (GSH), all inhibited (40–90%) covalent binding of radiolabel to liver and lung microsomes. The absence of O 2 (incubation under N 2) did not greatly affect the metabolism and covalent binding. Pretreatment of mice with various inducers, phenobarbital (PB),β-naphthoflavone (β-NF), pregnenolone 16α-carbonitrile (PCN) and 3-methylcholanthrene (3-MC), evoked increases in total liver microsomal cytochrome P-450 content (2-fold) and corresponding increases in covalent binding (3-fold). However, microsomes from PCN-treated mice showed only a 50% increase in DCE binding. Kidney microsomes from control, PB-, and β-NF-pretreated mice were incapable of covalent binding of radiolabel but those from PCN- and 3-MC-pretreated mice showed levels of binding similar to untreated mouse lung microsomes. It is proposed that the nephrotoxicity of DCE may be due to translocation of reactive metabolites from the liver to the kidney.

Metabolic activation of the serotonergic neurotoxin para-chloroamphetamine to chemically reactive intermediates by hepatic and brain microsomal preparations

Para-chloroamphetamine (PCA) is selectively toxic to serotonergie neurons in laboratory animals. Acute, reversible neurotoxicity is followed by long-term effects which include inactivation of tryptophan hydroxylase and destruction of neurons. We have studied the metabolic formation of reactive intermediates that might be involved in long-term PCA neurotoxicity. Incubation of [ 3H]PCA with rat hepatic microsomes resulted in NADPH-dependent and oxygen-dependent covalent binding of radioactivity to microsomal protein. Addition of SKF-525A and glutathione to incubation mixtures inhibited phjpca covalent binding 30% and 92% respectively. No inhibition of radiolabeled covalent binding was observed in an atmosphere of carbon monoxide/oxygen ( 80 20 ). 7,8-Benzoflavone was more effective than metyrapone in inhibiting [ 3H]PCA covalent binding. The extent of [ 3H]PCA covalent binding to microsomal protein was unchanged after phenobarbital pretreatment of rats, whereas 3-methylcholanthrene pretreatment increased [ 3H]PCA covalent binding (175%). NADPH-dependent and oxygen-dependent covalent binding of radioactivity was also observed when [ 3H]PCA was incubated with rat brain microsomal preparations. Addition of SKF-525A and glutathione to incubation mixtures inhibited covalent binding 10 and 40% respectively. There were no significant differences in total, NADPH-independent or NADPH-dependent covalent binding of radiolabeled R, S(±)-, R(-)-, or S(+)-PCA to rat hepatic microsomal protein. Less covalent binding was observed when [ 3H]amphetamine was incubated with rat liver microsomal preparations as compared to results with [ 3H]PCA. Minimal covalent binding was observed when [ 3H]PCA was incubated with liver microsomal preparations from rabbits, a species resistant to PCA neurotoxicity. Results of these metabolism studies are consistent with the hypothesis that oxidative metabolic activation of PCA to reactive and toxic metabolites is related to the long-term neurotoxicity of this agent.

NAD-Coupled Enzymatic Oxidation of O-Ethyl O-p-Nitrophenyl Phenylphosphonothioate (EPN) to Its Oxygen Analog with Liver Microsomes of Rats

O-Ethyl O-p-nitrophenyl phenylphosphonothioate (EPN)-induced inhibition of rat liver microsomal carboxylesterase (CEase) and formation of O-ethyl O-p-nitrophenyl phenylphosphonate (EPNoxon), an oxygen analog of EPN, were enhanced remarkably by addition of NAD in vitro. This potentiation of the anti-CEase action of EPN by NAD was significantly inhibited by addition of SKF 525-A or potassium thiocyanate (KSCN); and a simultaneous decrease in cytochrome P-450 contents was also observed. Addition of N-ethylmaleimide (NEM) at various concentrations inhibited potentiation of the anti-CEase action of EPN by NAD in parallel with inhibition of liver microsomal dehydrogenase activities. In conclusion, NAD was enzymatically reduced to NADH, a cofactor of microsomal dehydrogenase(s), and then formation of EPNoxon through microsomal cytochrome P-450-coupIed monooxygenase was accelerated. Consequently, inhibition of CEase by EPN was potentiated.

The role of glutathione and cytochrome P-450 in the metabolism of methyl chloride

Rat liver microsomes metabolized methyl chloride to formaldehyde at a rate 15-fold less than the rate of benzamphetamine demethylation. The reaction rate was stimulated approximately 2-fold in microsomes from phenobarbital-pretreated rats and was inhibited by addition of SKF-525A, carbon monoxide, metyrapone, and hexobarbital to the microsomal suspension, indicating dependence on cytochrome P-450. The in vivo incorporation of 14CH 3Cl into liver macromolecules, previously shown to reflect metabolism to CH 3Cl to formate, was not significantly altered by SKF-525A, Aroclor 1254, or 3-methylcholanthrene pretreatment of rats, although pretreatment with phenobarbital produced a 35 and 28% increase in 14CH 3Cl uptake into liver lipid and acid-insoluble material, respectively. Pretreatment with phenobarbital increased the in vivo metabolism of 14CH 3Cl to 14CO 2 (also derived from a formate intermediate) by 19%, but had no effect on urinary metabolites derived from 14CH 3Cl. SKF-525A inhibited 14CO 2 production from 14CH 3Cl by 30% and also had no effect on urinary excretion of 14C. In contrast, pretreatment with diethylmaleate inhibited 14CH 3Cl incorporation into liver macromolecules by 70 to 85%, and lowered 14CO 2 expiration and urinary 14C excretion by 52 and 60%, respectively. S-Methylcysteine pretreatment produced a similar inhibition of 14CH 3Cl incorporation and metabolism to 14CO 2; urinary excretion of 14C, however, was approximately doubled. 14CH 3Cl uptake into liver was also stimulated by cysteine pretreatment. These results indicate a strong dependence of CH 3Cl metabolism on tissue nonprotein sulfhydryl content and suggest a possible role for cytochrome P-450 in the in vivo metabolism of CH 3Cl. A scheme for the metabolism of CH 3Cl is postulated which involves initial reaction with glutathione, and sequential metabolism of the conjugate to S-methylcysteine, methanethiol, and formaldehyde.

Metabolic activation of hexamethylmelamine and pentamethylmelamine by liver microsomal preparations

Incubation of [ 14C]-ring labeled hexamethylmelamine and pentamethylmelamine with rat and mouse liver microsomal preparations results in metabolic activation of both drugs as measured by covalent binding of radiolabel to acid-precipitable microsomal macromolecules. Covalent binding is dependent on viable microsomes, NADPH, and molecular oxygen. Binding of HMM (280 pmol/mg protein/15 min) was approximately 5 times greater than that observed for PMM (60 pmol/mg protein/15 min), and represents 0.22% of incubated material. Similar results were found with [ 14C]-methyl labeled substrates. Pretreatment with phenobarbital increased covalent binding while addition of SKF 525-A, addition of glutathione, or incubation in an 80% carbon monoxide atmosphere reduced covalent binding.

Increased Aryl Hydrocarbon Hydroxylase Activity in Hepatic Microsomes from Streptozotocin-Diabetic Female Rats

In streptozotocin-induced diabetic male rats, hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity was depressed to less than control values, but was increased in microsomes from diabetic female rats. Insulin treatment of diabetic animals returned the altered AHH activity to control values in both sexes of rats. 2. Hepatic microsomal AHH activity was increased over control values in both sexes of diabetic mice. 3. Protection of female rats from the diabetogenic effects of streptozotocin by nicotinamide pretreatment also prevented the increase in AHH activity observed in unprotected animals. 4. Treatment of control and diabetic female rats with 3-methylcholanthrene resulted in larger increases in hepatic AHH activity in control animals, but similar increases in cytochrome P-448 content occurred in both treatment groups. 5. Differential stimulatory or inhibitory effects on AHH activity were observed after the addition of SKF 525-A, metyrapone, and rotenone to hepatic microsomes in vitro from control and diabetic female rats. However, similar stimulatory responses in AHH activity were observed after addition of alpha-naphthoflavone to microsomes from both treatment groups.

Metabolism of prostaglandin A1 by hepatic microsomal monooxygenase P-450 system in the guinea pig and rat.

Abstract This study demonstrates the metabolic transformation of prostaglandin A 1 (PGA 1 ) by guinea pig and rat liver microsomes. The transformation, which required NADPH and oxygen, yielded polar (presumably hydroxylated) products. Incubations with guinea pig liver microsomes yielded one zone of product on tlc, whereas rat liver microsomes produced two discernable metabolic zones. It was demonstrated that PGA 1 metabolism in the guinea pig and the rat was inhibited by the addition of SKF-525A, metyrapone, carbon monoxide and cytochrome C ; nicotinamide (10 mM) inhibited only the guinea pig system. These findings indicate that the enzymatic activity responsible for PGA 1 metabolism is composed of a typical cytochrome P-450 monooxygenase system.

The effect of diet on carbon tetrachloride metabolism

1. Blood and liver concentrations of carbon tetrachloride were measured, at intervals after an oral dose, in rats given stock and protein-free diets. The values did not correlate with the resistance to poisoning found in the rats on protein-free diets. 2. The metabolism of carbon tetrachloride to carbon dioxide in vivo and in liver microsomal preparations was depressed in animals given protein-free diets. 3. Rats given a single dose of DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] were highly sensitive to carbon tetrachloride poisoning. The livers of such animals had an increased microsomal protein content and greatly increased microsomal activity in the demethylation of Pyramidon (aminopyrine) and in the conversion of (14)CCl(4) into (14)CO(2). 4. The incorporation of [(14)C]leucine into protein by liver slices was depressed by carbon tetrachloride. This effect was decreased by addition of SKF525A (2-diethylaminoethyl 2,2-diphenyl-2-propylacetate) and in slices from rats given protein-free diets. It is suggested that the toxicity of carbon tetrachloride is closely linked to its metabolism.

Potentiation of cortisol induction of hepatic tyrosine transaminase by beta-diethylaminoethyl diphenylpropylacetate.

β-DIETHYLAMINOETHYL diphenylpropylacetate (SKF 525A) has been demonstrated in the rat to inhibit the metabolism of drugs both in vitro and in vivo1,2. We have demonstrated that the conversion of cortisol to a polar metabolite or metabolites by the 9,000g supernatant of the male rat liver is inhibited by the addition of SKF 525A to the incubation mixture3. The similarity between liver enzymes which metabolize drugs and those which hydroxylate steroids4 suggested the possibility that SKF 525A would inhibit the hydroxylation of cortisol in vivo. In turn, it was thought that this inhibition could manifest itself in an increase in the biological activity of administered cortisol. The administration of cortisol is known to increase the activity of rat liver tyrosine transaminase5,6. The present investigation demonstrates in the male rat a further increase in cortisol induction of tyrosine transaminase by SKF 525A.