Is there a beneficial effect of the calcium channel blocker diltiazem on cyclosporine A nephrotoxicity in rats?

To investigate whether or not there is a beneficial effect of diltiazem (D) on cyclosporine A (CsA) nephrotoxicity, renal function, CsA blood levels, and effects of CsA on biotransformation in the liver and on lipid peroxidation were characterized in rats. A single administration of D (60 mg/kg b.wt.) reduced urinary volume (UV), GFR and excretion of Na+ and K+, whereas a single dose of CsA (60 mg/kg b.wt.) alone had no respective effects. P-aminohippurate excretion was almost equal in all groups. Lower doses of D (and CsA) were without effects. After repeated CsA treatment a retardation in body weight gain was seen, with little effect of a co-administration with D hereon. In all tests, thymus mass was reduced by CsA, the weight of spleen, liver, adrenal glands, and kidney were not generally affected by any of the treatments. Furthermore, after repeated administration of CsA and/or D, urinary volume, GFR and Na+ excretion were reduced by CsA, too. Electrolyte concentrations in plasma showed no evident changes by any of the treatments for Na+ and Ca2+. After long time treatment, CsA and CsA + D quite similarly led to higher K+ but lower Mg2+ concentrations in plasma. Only with 7 days highest dosage treatment PAH excretion was reduced significantly by CsA and CsA + D treatment. Surprisingly, CsA levels measured in blood and in kidney tissue, showed lower values after co-administration with D compared to CsA treatment alone. This could be caused by higher activities of monooxygenase functions revealed after pretreatment with D alone. Reduced glutathione (GSH) contents in kidney were elevated in CsA and CsA + D treated groups. In general no significant differences were to be observed concerning lipid peroxidation and stimulated H2O2 formation. Altogether evident protective effects of diltiazem on CsA nephrotoxicity in rats could not be proven.

Cytochrome P450-dependent drug oxidation activities in liver microsomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans.

Levels of cytochrome P450 (P450 or CYP) proteins immunoreactive to antibodies raised against human CYP1A2, 2A6, 2C9, 2E1, and 3A4, monkey CYP2B17, and rat CYP2D1 were determined in liver microsomes of rats, guinea pigs, dogs, monkeys, and humans. We also examined several drug oxidation activities catalyzed by liver microsomes of these animal species using eleven P450 substrates such as phenacetin, coumarin, pentoxyresorufin, phenytoin, S-mephenytoin, bufuralol, aniline, benzphetamine, ethylmorphine, erythromycin, and nifedipine; the activities were compared with the levels of individual P450 enzymes. Monkey liver P450 proteins were found to have relatively similar immunochemical properties by immunoblotting analysis to the human enzymes, which belong to the same P450 gene families. Mean catalytic activities (on basis of mg microsomal protein) of P450-dependent drug oxidations with eleven substrates were higher in liver microsomes of monkeys than of humans, except that humans showed much higher activities for aniline p-hydroxylation than those catalyzed by monkeys. However, when the catalytic activities of liver microsomes of monkeys and humans were compared on the basis of nmol of P450, both species gave relatively similar rates towards the oxidation of phenacetin, coumarin, pentoxyresorufin, phenytoin, mephenytoin, benzphetamine, ethylmorphine, erythromycin, and nifedipine, while the aniline p-hydroxylation was higher and bufuralol 1'-hydroxylation was lower in humans than monkeys. On the other hand, the immunochemical properties of P450 proteins and the activities of P450-dependent drug oxidation reactions in dogs, guinea pigs, and rats were somewhat different from those of monkeys and humans; the differences in these animal species varied with the P450 enzymes examined and the substrates used. The results presented in this study provide useful information towards species-related differences in susceptibilities of various animal species regarding actions and toxicities of drugs and xenobiotic chemicals.

Differential effects of neuroleptic agents on hepatic cytochrome P-450 isozymes in the male rat.

We report the effects of various dopamine receptor-blocking drugs on gene and protein expression, as well as the activity of several hepatic cytochrome P-450 (CYP) enzymes in the male Sprague-Dawley rat. At equipotent doses (with respect to receptor blockade and behavioural tests), the dopamine D2-receptor selective sulpiride and remoxipride gave a conspicuous down-regulation of CYP2C11 and its associated androstenedione 16 alpha-hydroxylation activity as well as of the CYP2C11-specific mRNA. The average immunoidentified CYP2C11 levels correlated with the CYP2C11-specific mRNA levels in all treatment groups (r = 0.994), indicating a transcriptional mechanism. The CYP3A protein was also selectively down-regulated. In contrast, androstenedione 5 alpha-reduction was significantly increased. Clozapine, a non-selective neuroleptic, gave the same effects on the steroid metabolism as sulpiride and remoxipride. In contrast, diverging effects were observed for clozapine, compared to sulpiride and remoxipride, on the immunoidentified CYP1A2, CYP2B1, and CYP3A. These proteins were elevated by clozapine, and down-regulated by sulpiride and remoxipride. Our results are of interest for the interpretation of preclinical dose ranging toxicity tests of neuroleptic agents in rats. They may also be relevant in relation to certain interactions and adverse reactions observed in the clinical use of these drugs. The down-regulation of certain CYP enzymes is most likely mediated by an interaction with the growth hormone secretion.

Hepatic cytochrome P450 induction in goats. Effects of model inducers on the metabolism of alkoxyresorufins, testosterone and ethylmorphine, and on apoprotein and mRNA levels.

Male and female African dwarf goats were treated orally with phenobarbital (PB) or triacetyloleandomycin (TAO), or subcutaneously with beta-naphthoflavone (BNF). Hepatic microsomal cytochrome P450 content was increased by PB and TAO, but not by BNF. PB effects on P450 activities were non-selective: ethoxyresorufin deethylase (EROD) and pentoxyresorufin depentylase (PROD), hydroxylation of testosterone (TST) and demethylation of ethylmorphine (ETM) were all induced by a factor of 2-3. A similar non-selective induction was observed with TAO, except for EROD and PROD (no effects). After PB and TAO treatment, increased levels of a protein cross-reactive with anti-sheep P450 3A and 2B were found. Thus, in dwarf goats, both PB and TAO appeared to be P450 3A inducers. Selective PB effects related to a P450 2B form on PROD are lacking but 16 alpha-hydroxylation of TST was induced markedly. At the mRNA level, PB induced an mRNA that showed good sequence homology with a human P450 3A4 cDNA probe, rather than with a rat 3A1 probe. BNF selectively induced EROD, whereas TST hydroxylation and ETM dealkylation were inhibited. With BNF-treated animals, increased concentrations of a protein cross-reactive with anti-rat P450 1A1/1A2 and of an mRNA that showed homology with a human 1A1 cDNA probe, but not with a mouse 1A1/1A2 probe, were observed.

N-demethylation of ethylmorphine in pregnant and non-pregnant women and in men: an evaluation of the effects of sex steroids.

1. The effects of oestrogens, testosterone, progesterone and medroxyprogesterone acetate (MPA) on the rate of N-demethylation of ethylmorphine (EM) to norethylmorphine (NEM) were studied in human adult liver microsomes. 2. N-Demethylase activity was found to be inhibited by progesterone and MPA to a similar extent while oestrogens and testosterone had no or negligible effects. 3. These findings prompted us to measure the N-demethylation of EM in relation to serum progesterone concentration in vivo in three groups of volunteers with large physiological differences in their endogenous levels of progesterone, i.e. i) pregnant women, ii) non-pregnant ovulating women and iii) men. 4. The metabolic ratio (MRP) of EM to NEM in plasma 60 min after dosage and the corresponding ratio in urine sampled for 6 h (MRU,1), measured on two occasions 14 days apart were used to reflect intraindividual variation in the rate of N-demethylation. 5. The average difference in MRP and MRU,1 between the two occasions was similar in all groups. However, the variability in MRP between individuals within a group was significantly higher in ovulating women than in men, but this had no relation to the serum concentrations of progesterone or oestradiol. 6. The cumulative 12 h urinary excretion of EM, NEM and morphine (MO) after hydrolysis with beta-glucuronidase was about 46%. There was no difference in the metabolic ratio of EM to NEM and its conjugate(s) in the urine between the luteal and the follicular phases. Our findings suggest that the menstrual cycle does not influence the rate of N-demethylation of EM.

Effect of the position of the cyano-group of cyanopregnenolones on their drug metabolic inducing activity.

The effect of the position of the cyano-group of several cyanopregnenolones on the body's resistance to drugs and on drug metabolism was investigated. Female rats were pretreated with 2 alpha-, 6-, 16 alpha-, 17 alpha-cyano- or 16 alpha-cyanomethyl-pregnenolone or with pregnenolone, and the (in vivo) resistance to zoxazolamine, digitoxin and indomethacin, as well as the in vitro drug metabolism (post mitochondrial fraction) of zoxazolamine and ethylmorphine were determined. It was found that the 16-derivative was the most active in this respect, the 2- and 17-cyanopregnenolones were less active but significantly potent compared to controls, while the 6-cyano, the 16-cyanomethyl derivatives and pregnenolone were essentially inactive. These differences were explained in terms of an effective or poor fit of the steroids to their receptor. The poor performance of pregnenolone-16 alpha-acetonitrile was attributed to electronic effects. A hypothesis of some structural features of the receptor site for its interaction with the cyanopregnenolone inducers was presented.

Effects of cage beddings on microsomal oxidative enzymes in rat liver.

The purpose of the present studies was to evaluate the effects of some commercially available cage beddings on rat liver microsomal cytochrome P-450-dependent drug-metabolizing enzyme, ethylmorphine N-demethylase, and the carcinogen-metabolizing enzyme, benzo(a)pyrene hydroxylase. Sprague-Dawley rats were housed in cages containing cedar chip, corncob or heat-treated pinewood bedding for 3 weeks. Control rats were housed in cages on wire bottom floors containing no bedding material. Rats housed in cages containing cedar chip showed 18, 46 and 49% increases in liver cytochrome P-450 content, ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase activities, respectively. The liver enzyme activities of rats housed in cages containing corncob bedding were similar to those obtained with control rats. In contrast, the pinewood-bedded rats showed a 21% decrease in ethylmorphine N-demethylase activity without affecting cytochrome P-450 content and benzo(a)pyrene hydroxylase activity. Hexobarbital-induced sleep times of the variously bedded rats were similar to those of control animals. These data suggest that the commercial bedding materials differ in their abilities to affect liver microsomal enzymes. Thus, interlaboratory variability in basal enzyme activities reported in the literature may be partly due to bedding materials used in the animal's cages.

Dantrolene sodium: urinary metabolites and hepatotoxicity.

Dantrolene is metabolized by the hepatic MFO system to hydroxydantrolene which is conjugated with glucuronic acid or with sulfate. Dantrolene is also metabolized by nitroreductase to aminodantrolene. The aminodantrolene inhibits the hepatic MFO system. Acetylation of aminodantrolene blocks the inhibitory effects of aminodantrolene on the hepatic MFO system. Intermediates in the nitroreductase pathway form glucuronide and mercapturic acid conjugates. The mercapturic acid conjugation reaction is a detoxification mechanism for an electrophilic metabolite of dantrolene.

Influence of dietary thiamin on phenobarbital induction of rat hepatic enzymes responsible for metabolizing drugs and carcinogens.

Male and female Sprague-Dawley rats were fed synthetic diets deficient in or supplemented with thiamin for 2 or 3 weeks. One group of rats receiving the thiamin-supplemented diet was pair-fed the amount consumed by rats fed the thiamin-deficient diet. One-half of each group was administered phenobarbital sodium for four consecutive days prior to decapitation. Rats fed the thiamin-deficient diet had higher NADPH cytochrome c reductase, aniline hydroxylase, and ethylmorphine N-demethylase activities than those fed high levels of thiamin. In addition, these animals generally responded more vigorously to induction by phenobarbital in their synthesis of microsomal protein, and increased activities of NADPH cytochrome c reductase, aniline hydroxylase, and ethylmorphine N-demethylase. Cytochrome P-450 concentration was higher in the microsomes from thiamin-deficient rats and was induced to a greater degree by phenobarbital than in microsomes from rats fed thiamin-supplemented diets ad libitum. Phenobarbital-enhanced metabolism of N-nitrosodimethylamine (DMN) by liver 9,000 g supernatant as evidenced by approximately two-fold increases in formaldehyde formed per gram liver. This increase in DMN metabolism in male rats is due at least in part to the increased concentration of microsomal protein, since metabolism per milligram microsomal protein was not increased. The fact that DMN metabolism per unit of microsomal cytochrome P-450 in phenobarbital-treated animals is decreased to about one-half of that in controls indicates that DMN is either metabolized by a non-cytochrome P-450-dependent system or that it is metabolized by a form of P-450 not induced by phenobarbital. A sex difference was evident in these experiments, females generally being more sensitive to the influence of varying levels of dietary thiamin. Also female rats but not males fed high-thiamin diets responded to phenobarbital with increased DMN metabolism per milligram microsomal protein.

Alterations of microsomal monooxygenase system and carcinogen metabolism by streptozotocin-induced diabetes in rats.

The effects of diabetes on the liver microsomal monooxygenase enzymes and carcinogen metabolism have been studied in rats. Treatment with streptozotocin causes a marked enhancement in microsomal N-nitrosodimethylamine (NDMA) demethylase activity. The enhancement is due mainly to the induction of a high affinity NDMA demethylase (Km, approximately 0.05 mM) which is accompanied by the induction of a protein species with mol. wt. of 50,000. The treatment also induces aniline hydroxylase whose activity is in parallel with NDMA demethylase. Streptozotocin-induced diabetes also increases the metabolism of N-nitrosomethylethylamine but not that of N-nitrosomethylaniline or N-nitrosomethylbenzylamine. On the other hand, diabetes decreases the metabolism of benzo[a]pyrene, benzphetamine, and ethylmorphine. The result suggest that diabetes causes an alteration of the composition of cytochrome P-450 isozymes; the forms efficient in metabolizing NDMA are increased while certain other forms of cytochrome P-450 are decreased.

Studies on cadmium-induced inhibition of hepatic microsomal drug biotransformation in the rat.

Cadmium is a potent inhibitor of hepatic microsomal drug biotransformation in the rat. Male rats receiving a single intraperitoneal dose of cadmium exhibit significant decreases in hepatic microsomal metabolism of a variety of substrates. The threshold cadmium dose is 0.84 mg Cd/kg, and the effect lasts at least 28 days. Mechanistically, the inhibitory effect results from decreased cytochrome P-450 content since cadmium does not alter NADPH cytochrome c reductase activity. This effect is also observed following acute oral administration of cadmium in doses greater than 80 mg Cd/kg but is not observed following chronic administration of the metal via drinking water in concentrations of 5-200 ppm for periods ranging from 2 to 50 weeks. A tolerance to the inhibitory cadmium effect is observed if male rats are pretreated with subthreshold doses of the metal prior to the challenge cadmium dose. The degree of tolerance can be overcome by increasing the challenge dose of cadmium. Characterization of the tolerance phenomenon in terms of onset, duration, and intensity reveals a good correlation with the kinetics of metallothionein production, suggesting that the underlying basis for the tolerance phenomenon is likely the induction of metallothionein. A sex-related difference in the inhibitory effect of cadmium was observed. Cadmium did not inhibit the metabolism of hexobarbital or ethylmorphine in female rats but did inhibit that of aniline or zoxazolamine. Cadmium did not lower cytochrome P-450 content in female rats.