Induction of intestinal microsomal enzymes by polycyclic aromatic hydrocarbons.

1. The efficacy of induction of intestinal microsomal aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin o-deethylase and cytochrome P-450 by various polycyclic aromatic hydrocarbons were studied. 2. The greatest induction of the specific MFO activities tested was produced by 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) with 3-methyl-cholanthrene (MC) a close second. 3. Rank order of efficacy of induction of spectrally determined cytochrome P-450 was 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), Arochlor 1254 (ARO), 3-methylcholanthrene (MC) and benz(a)anthracene(BA). 4. No unique protein peaks were found when the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MC treated intestinal microsomes were prepared.

Dietary iron and 2,3,7,8-tetrachlorodibenzo-p-dioxin induced alterations in hepatic lipid peroxidation, glutathione content and body weight.

The effects of various levels of dietary iron on hepatic lipid peroxidation (malondialdehyde [MDA] content), reduced glutathione (GSH) and GSH peroxidase (GSH-PX) activity as well as liver and body weights of female rats following TCDD administration were examined. Rats were fed diets containing deficient (6 ppm), normal (35 ppm) and supplemented (120 ppm) iron for 17, 24 and 31 days. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, 40 micrograms/kg/day P.O.) in corn oil or the vehicle was given on days 9, 8 and 7 prior to sacrifice. TCDD treatment produced a 3-fold increase in hepatic MDA content in animals on normal iron diet. TCDD administration failed to increased MDA content in iron deficient animals. In the iron supplemented groups, TCDD resulted in 2.5 fold increases in lipid peroxidation. Dietary iron had no effect on hepatic GSH-PX activity. Animals on the iron deficient diet had 12-21% decreases in hepatic GSH content. TCDD administration resulted in 15-22% decreases in GSH content in animals on the control and iron supplemented diets. TCDD treatment resulted in significant decreases in body weights of animals on all 3 diets. TCDD induced lipid peroxidation appears to be iron dependent. However, the loss in body weight due to TCDD toxicity may not be dependent on lipid peroxidation.

Influence of protein and RNA synthesis inhibitors on methylcholanthrene-mediated induction of aryl hydrocarbon hydroxylase activity in intestinal mucosa.

The sensitivity of the 3-methylcholanthrene (MC)-mediated induction of aryl hydrocarbon hydroxylase (AHH) activity to pretreatment with inhibitors of protein and RNA synthesis was studied. Cordycepin significantly inhibited the induction at 12 h but not at 3 h post-treatment with MC. Both cycloheximide and actinomycin D significantly inhibited the MC-mediated induction at 3 and 12 h after MC treatment. An MC-mediated stimulation of 3H-leucine incorporation into microsomal protein in vivo was observed at 12 h following MC treatment. A cell-free protein synthesis system employing intestinal ribosomes was developed. The rate of protein synthesis (3H-leucine incorporation/mg rRNA/3 min) and the number of active ribosomes (3H-peptidyl-puromycin formed/mg rRNA) were determined. No effect of in vivo MC treatment was observed in subsequently isolated intestinal ribosomes at either 3, 9 or 18 h post-treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on intestinal microsomes prepared from control, corn-oil- and MC-treated rats 18 h after treatment. The microsomal proteins depicted only slight quantitative changes in microsomal proteins following MC treatment, and no unique protein peaks. These results may explain the lack of detectable stimulation of the activity of isolated ribosomes. In addition, the methods used may lack sufficient sensitivity to detect the small net change in protein necessary to account for the increase in AHH activity seen after MC induction.

Effect of cimetidine on the pharmacokinetics of theophylline.

1. The effect of the therapeutic doses of cimetidine (400 mg/twice daily) on theophylline metabolism in Jordanian volunteers was studied. 2. The administration of the above therapeutic cimetidine dose did not alter theophylline clearance and elimination half-life. 3. Cimetidine administration also failed to alter the elimination of theophylline metabolites in urine.

Effect of ofloxacin on the pharmacokinetics of a single intravenous theophylline dose.

The effect of ofloxacin taken for 8 days (200 mg twice daily) on the pharmacokinetics of a single intravenous dose of theophylline (4.3 mg/kg over 15 min) was studied in a crossover procedure among seven healthy male volunteers. Theophylline concentrations were measured serially for 10 h by the immunofluorescence polarization technique. No significant effect of ofloxacin was found on theophylline clearance, half-life, or volume of distribution. It is therefore concluded that ofloxacin and theophylline can be safely administered together.

Spectrophotometric study of the photodecomposition kinetics of nifedipine.

Nifedipine is a photosensitive compound. Irradiation for 4 h under a fluorescent lamp placed 30 cm from a solution of nifedipine in 95% ethanol leads to complete photo-oxidation as determined spectrophotometrically. The disappearance of the reduced form and appearance of the oxidized form is best described by zero-order kinetics at concentrations higher than 4 x 10(-4) M. At lower concentrations pseudo-first order kinetics are followed. Monochromatic irradiation of nifedipine at wavelengths 400 to 700 nm in 25 nm increments showed no change in the absorbance at 280 nm, and, except for a hyperchromic effect at 237 nm, no other spectral changes were observed. Its photo-oxidation was dependent on the intensity of light and increased exponentially as solutions were irradiated progressively closer to a fluorescent light source. The pH studies showed that aqueous solutions of nifedipine photo-oxidized fastest at pH 2.

Comparative determination of phenytoin in plasma by fluorescence polarization immunoassay and high performance liquid chromatography.

The need for careful monitoring of plasma concentrations of phenytoin during use of the drug in the treatment of epilepsy is well recognized; there can be great intersubject variation in the absorption rate and clearance rate of the drug, and its therapeutic ratio is narrow. In this study, two methods for determining plasma phenytoin concentrations were compared. One, based on fluorescence polarisation immunoassay (FPIA), is utilised in a commercially-available kit. The other, our own modification of a published procedure, was based on high performance liquid chromatography (HPLC). The accuracy and precision of both methods were evaluated, and the coefficients of variation (C.V.) were calculated. The C.V.'s ranged from 0.71 to 1.86% for the FPIA method, and from 2.81 to 8.69% for the HPLC method. Corresponding bias values were 1.20 to 1.60%, and 2.81 to 8.69%, respectively. A good correlation coefficient (0.977) was obtained, but estimated phenytoin concentrations were significantly higher (95% confidence level) using the HPLC method. We conclude that both methods perform adequately for clinical purposes. The HPLC method is, however, less expensive than the FPIA method.

Effects of glucagon on cAMP accumulation and ketogenesis in hepatocytes from euthyroid and hypothyroid rats.

The resistance to the effects of glucagon was studied in isolated hepatocytes prepared from male rats treated with 6N-propyl-2-thiouracil (PTU). Incorporation of [14C]oleate into ketone bodies in response to various concentrations of glucagon (10(-5) to 10(-10) M) was reduced in hepatocytes from hypothyroid rats compared with the euthyroid group. The reduced sensitivity to the effects of glucagon on ketogenesis after treatment with PTU was associated with a reduced ability of those hepatocytes to maintain cyclic adenosine-3',5'-monophosphate (cAMP) at levels required to stimulate ketogenesis. The concentration of cAMP in response to glucagon (10(-5) to 10(-10) M) was diminished in hepatocytes from hypothyroid rats, compared with those from euthyroid animals.

Glutathione, glutathione S-transferase and glutathione reductase in human erythrocytes and lymphocytes as a function of sex.

We review the role of glutathione (GSH) and its metabolizing enzymes, glutathione S-transferase (GST) and glutathione reductase (GSR) in drug metabolism and in the elimination of foreign compounds. Levels of GSH and the activity of these enzymes may be greatly influenced by drugs and other substances in the body. We therefore determined GSH levels and the activities of GST and GSR in human erythrocytes and lymphocytes in males and females in three age groups. There was no significant difference between males and females in the three age groups in respect of GSH levels and GST and GSR activities. GSH levels in erythrocytes were higher than those in lymphocytes when expressed per mg protein, but lower than those in lymphocytes when expressed per 10(6) cells. The activities of both GST and GSR were found to be higher in lymphocytes than in erythrocytes.

Changes in glutathione, glutathione reductase and glutathione-S-transferase as a function of cell concentration and age.

The activities of glutathione-S-transferase (GST) and glutathione reductase (GSR) in mouse lymphocytes as a function of cell concentration and age were determined. Lymphocytes from 2-, 9- and 24-month-old mice were isolated and the activity of GST was determined spectrophotometrically using 1-chloro-2,4-dinitrobenzene as the substrate. Lymphocyte concentrations of 0.44, 0.75 and 0.55 X 10(6) cells/ml were found to be optimal for assaying GST in 2-, 9- and 24-month-old mice, respectively. Determination of GSR activity was based on NADPH reduction of oxidized glutathione and cell concentrations of 0.29, 0.30 and 0.22 X 10(6) cells/ml were chosen for assaying the enzyme activity for the three age groups, respectively. Glutathione levels and GSR activity of mice lymphocytes were higher in 2- and 24-month-old mice as compared to 9-month-old animals. However, GST activity of mouse lymphocytes was low in 2- and 9-month-old mice and increased significantly in 24-month-old animals.

Changes in glutathione and its metabolizing enzymes in human erythrocytes and lymphocytes with age.

The levels of glutathione (GSH) and the activities of glutathione S-transferases (GST) and glutathione reductase (GSR) in human erythrocytes and lymphocytes were determined in three age groups (5-12, 25-40, 65-83 years). The levels of GSH in lymphocytes increased with age, however, its levels in erythrocytes reached a maximum in the middle age group. The activity of GST in erythrocytes and lymphocytes changed as a function of age in a pattern similar to the changes found for GSH levels. GSR activity increased from young to middle age in both erythrocytes and lymphocytes, but decreased again in the old age group.

Effect of forskolin on cAMP accumulation and ketogenesis in isolated hepatocytes.

The effect of forskolin on ketogenesis and cAMP accumulation was studied in hepatocytes from euthyroid and hypothyroid rats. Forskolin stimulated ketogenesis, cAMP production and potentiated glucagon-stimulated cAMP accumulation on both euthyroid and hypothyroid groups. The ketogenic effect of glucagon was inhibited by forskolin in both groups. Also, forskolin, glucagon and methylisobutylxanthine (MIX) combined did not significantly increase ketogenesis.

Glutathione, glutathione reductase and glutathione S-transferase activities in erythrocytes and lymphocytes in chronic renal disease.

Erythrocyte and lymphocyte reduced glutathione (GSH) levels, and glutathione reductase (GR) and glutathione S-transferase (GST) activities have been investigated in uremic patients pre- and post-dialysis and normal subjects of the same age span. GSH levels and GST activities in erythrocytes and lymphocytes and GR activity in lymphocytes from uremic patients were higher as compared to the corresponding controls. Dialysis resulted in a 17% decrease in GSH levels in erythrocytes. Hemodialysis did not significantly alter erythrocyte GST and GR activities. Hemodialysis produced a 50% decrease in lymphocyte GST activity and a 32% decrease in lymphocyte GR activity. Elevated levels of GSH and increased activities of GST and GR in blood cells of uremic patients may be associated with a compensatory protective mechanism against accumulating toxic wastes in uremic plasma.

Changes in glutathione and glutathione metabolizing enzymes in erythrocytes and lymphocytes of mice as a function of age.

Changes in reduced glutathione levels in liver, lung and whole blood of female Swiss-Webster mice with age were determined. In addition, glutathione content, and glutathione S-transferase and glutathione reductase activities of erythrocytes and lymphocytes of mice as a function of age were examined. Reduced glutathione content increased in liver, lung, whole blood, erythrocytes and lymphocytes with age from 3 to 9 months, reached a maximum level at 9 months of age and decreased thereafter with advanced age in all tissues. Glutathione S-transferase and glutathione reductase activities in erythrocytes and lymphocytes increased with age from 3 to 9 months, reached maximum activities at 9 months and decreased thereafter with advanced age. The glutathione content of erythrocytes from animals 18 months of age decreased by 56% as compared to 9 month old mice, while the activities of glutathione S-transferase and glutathione reductase decreased by 56 and 48%, respectively, over the same age span.

Diminished conjugation of products of mixed-function oxidation in perfused livers from hypophysectomized rats.

The rates of metabolism of 7-ethoxycoumarin and subsequent conjugation of 7-hydroxycoumarin were studied in perfused livers from hypophysectomized or sham-operated control rats. Rates of 7-ethoxycoumarin O-deethylation were higher both in perfused livers and microsomes from hypophysectomized compared to sham-operated controls. However, conjugation of 7-hydroxycoumarin formed from 7-ethoxycoumarin was markedly decreased by hypophysectomy. p-Nitrophenol conjugation was also impaired in perfused livers from hypophysectomized rats. During infusion of 59-65 microM of p-nitrophenol into hypophysectomized livers, rates of glucuronidation were diminished by 45% (6.83 +/- 0.49 to 3.78 +/- 0.31 mumol/g/h) and rates of sulfation were decreased by 50% (1.25 +/- 0.12 to 0.62 +/- 0.07 mumol/g/h). Phenol sulfotransferase activity was decreased 57% by hypophysectomy, which is the likely explanation for the decrease in sulfate conjugation. However, hypophysectomy did not affect glucuronyltransferase activity. Perfused livers from hypophysectomized rats released glucose, pyruvate and lactate at lower rates than livers from sham-operated rats. Furthermore, infusion of glucose into perfused livers from hypophysectomized rats, but not sham-operated controls, increased the rate of conjugation. The results demonstrate that hypophysectomy decreased rates of conjugation in perfused livers, and that the decreased glucuronidation rates are probably the result of diminished carbohydrate reserves.

In vitro screening of the effect of hydrazine derivatives on hepatic aryl hydrocarbon hydroxylase activity.

1. The effect of hydrazine derivatives on the in vitro hepatic microsomal aryl hydrocarbon hydroxylase activity was examined using Sprague-Dawley male rats. 2. All hydrazine derivatives examined have some degree of inhibition on hepatic aryl hydrocarbon hydroxylase activity. 3. The inhibitory effect is concentration dependent and is also dependent on the structural substitution of the hydrazines. The more lipophilic the derivative, the more potent the inhibition of aryl hydrocarbon hydroxylase activity.

Kinetics of tamoxifen inhibition of aryl hydrocarbon hydroxylase activity of intestinal and hepatic microsomes from male rats.

The in vitro effects of tamoxifen on microsomal aryl hydrocarbon hydroxylase (AHH) activity from male rat liver and intestinal mucosa were examined. Tamoxifen inhibited microsomal activity from both liver and intestinal mucosa. Tamoxifen was a much more potent inhibitor of hepatic AHH activity as compared to its effects on intestinal AHH activity. At 0.50 mM, tamoxifen inhibited approximately 92% of AHH activity and only 7% of the intestinal activity. Kinetic studies demonstrated that the inhibition of hepatic AHH activity by tamoxifen at concentrations of 0.10 mM or less was non-competitive. However, the same tamoxifen concentrations gave an uncompetitive inhibition when intestinal microsomal AHH activity was studied. The data indicates that AHH inhibition by tamoxifen is both tissue and concentration dependent.

Glutathione S-transferase activity in liver, lung and intestinal mucosa of aging female mice.

1. Changes in the activity of glutathione S-transferase (GST) in liver, lungs and intestinal mucosa of female Swiss-Webster mice with age were determined. 2. GST activity increased with age from 1 to 9 months, reached a maximum activity at 9 months and decreased thereafter with advanced age in all three tissues. 3. GST activity decreased by approximately 75% in liver between 9 and 18 months, with decreases of 65 and 64% occurring over the same time period for lung and intestinal mucosa, respectively.

Effect of tamoxifen treatment on liver, lung and intestinal mixed-function oxidases in male and female rats.

Tamoxifen is a nonsteroidal antiestrogen which is used as an adjuvant form of chemotherapy for breast carcinomas containing estrogen receptors. Tamoxifen citrate (2 mg/rat/day) administration to male rats significantly decreased hepatic microsomal aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-de-ethylase activities and cytochrome P-450 content. These effects may be exerted through an antiandrogenic activity of tamoxifen, because plasma testosterone concentrations were also decreased. In male rats, tamoxifen treatment also depressed lung and intestinal microsomal aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-de-ethylase activities. Tamoxifen citrate treatment of female rats had no effect on hepatic, pulmonary, or intestinal microsomal aryl hydrocarbon hydroxylase or 7-ethoxycoumarin O-de-ethylase activities or hepatic cytochrome P-450 content. The results support the contention that estrogens at physiologic levels do not exert a significant regulatory effect on xenobiotic metabolism. Furthermore, androgens are known to influence drug metabolism, and the results indicate that tamoxifen has some antiandrogenic activity.