Zoxazolamine-induced stimulation of cardiomyogenesis from embryonic stem cells is mediated by Ca2+, nitric oxide and ATP release.

Ca2+-activated potassium (KCa) channels of small and intermediate conductance influence proliferation, apoptosis, and cell metabolism. We analysed whether prolonged activation of KCa channels by zoxazolamine (ZOX) induces differentiation of mouse embryonic stem (ES) cells towards cardiomyocytes. ZOX treatment of ES cells dose-dependent increased the number and diameter of cardiac foci, the frequency of contractions as well as mRNA expression of the cardiac transcription factor Nkx-2.5, the cardiac markers cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), ventricular myosin light chain-2 (MLC2v), and the pacemaker hyperpolarization-activated, cyclic nucleotide-gated 4 channel (HCN4). ZOX induced hyperpolarization of membrane potential due to activation of IKCa, raised intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) in a Ca2+-dependent manner. The Ca2+ response to ZOX was inhibited by chelation of Ca2+ with BAPTA-AM, release of Ca2+ from intracellular stores by thapsigargin and the phospholipase C (PLC) antagonist U73,122. Moreover, the ZOX-induced Ca2+ response was blunted by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) as well as the specific P2Y1 antagonist MRS 2,179, suggesting purinergic receptor-stimulated signal transduction. Consequently, ZOX initiated ATP release from differentiating ES cells, which was inhibited by the chloride channel inhibitor NPPB and the gap junction inhibitor carbenoxolone (CBX). The stimulation of cardiomyogenesis by ZOX was blunted by the nitric oxide synthase (NOS) inhibitor l-NAME, as well as CBX and NPPB. In summary, our data suggest that ZOX enhances cardiomyogenesis of ES cells by ATP release presumably through gap junctional hemichannels, purinergic receptor activation and intracellular Ca2+ response, thus promoting NO generation.

Acute and Subchronic Toxicity Studies of Aqueous Extract of Desmodium adscendens (Sw) DC.

Extracts of Desmodium adscendens (Sw) DC are used for the treatment of various diseases but limited toxicological evaluations have been done on the medicinal plant. This study investigates toxicity effects of the leave extract of D adscendens, and the possibility of drug-drug interaction of the plant extract when co-administered with other drugs. Oral administrations of leaf extract of D adscendens to white Wistar rats in an acute toxicity studies allowed the estimation of an LD50 (median lethal dose) value of 1122 mg/kg body weight. In a subchronic toxicity studies, the plant extract caused a decrease in zoxazolamine paralysis time and prevented thiopentone from causing sleep in test animals compared to controls. Overall, the results are consistent with the plant extract being safe at the doses administered in humans. However, the induction of the CYP enzymes is an indication of a possible drug interaction when the plant extract is co-administered with other drugs.

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters.

AIM: Our aim is to investigate the molecular mechanism of regulation of gene expression of drug metabolizing enzymes (DMEs) and transporters in diet-induced obesity. MAIN METHODS: Adult male CD1 mice were fed diets containing 60% kcal fat (HFD) or 10% kcal fat (LFD) for 14 weeks. RNA levels of hepatic DMEs, transporters and their regulatory nuclear receptors (NRs) were analyzed by real-time PCR. Activation of cell-signaling components (JNK and NF-κΒ) and pro-inflammatory cytokines (IL-1ß, IL-6 and TNFα) were measured in the liver. Finally, the pharmacodynamics of drugs metabolized by DMEs was measured to determine the clinical relevance of our findings. KEY FINDINGS: RNA levels of the hepatic phase I (Cyp3a11, Cyp2b10, Cyp2a4) and phase II (Ugt1a1, Sult1a1, Sultn) enzymes were reduced ~30-60% in HFD compared to LFD mice. RNA levels of Cyp2e1, Cyp1a2 and the drug transporters, multidrug resistance proteins, (Mrp)2, Mrp3 and multidrug resistant gene (Mdr)1b were unaltered in HFD mice. Gene expression of the NRs, PXR and CAR and nuclear protein levels of RXRα was reduced in HFD mice. Cytokines, JNK and NF-κΒ were induced in HFD mice. Thus reduction in hepatic gene expression in obesity may be modulated by cross-talk between NRs and inflammation-induced cell-signaling. Sleep time of Midazolam (Cyp3a substrate) was prolonged in HFD mice, while Zoxazolamine (Cyp1a2 and Cyp2e1 substrate)-induced sleep time was unaltered. SIGNIFICANCE: This study demonstrates that gene-specific reductions in DMEs can affect specific drugs metabolized by these enzymes, thus providing a rationale to monitor the effectiveness of drug therapy in obese individuals.

Inhibitory effect of magnolol on Trp-P-2-induced DNA damage in various organs in mice.

Magnolol has been reported to strongly inhibit the mutagenicity induced by indirect mutagens in the Ames test as well as the clastogenicity induced by benzo(a)pyrene (B(a)P) in the mice micronucleus test. Here, we evaluated the inhibitory effect of magnolol on the DNA damage induced by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in various organs using the mice alkaline single cell gel electrophoresis (SCG) assay. Animals were treated with a single oral administration of magnolol (0.01, 0.1, 1, 10, and 100 mg/kg), followed by a single intraperitoneal injection of Trp-P-2 (10 mg/kg). The liver, lung, and kidney were removed at 3 h after treatment and used in SCG assay. The results indicated that magnolol inhibited Trp-P-2-induced DNA damage in various organs. To elucidate the mechanism of this inhibitory effect against Trp-P-2, we investigated the inhibitory effect of magnolol on in vivo CYP1A2 activity using the zoxazolamine paralysis test. Magnolol significantly prolonged zoxazolamine paralysis time and showed an inhibitory effect on in vivo CYP1A2 activity. These results indicate that magnolol has an inhibitory effect on the DNA damage induced by Trp-P-2 in various organs in vivo. This inhibitory mechanism is considered due to in vivo CYP1A2 inhibition.

Sexually dimorphic regulation and induction of P450s by the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR) is a xenosensing nuclear receptor and regulator of cytochrome P450s (CYPs). However, the role of CAR as a basal regulator of CYP expression nor its role in sexually dimorphic responses have been thoroughly studied. We investigated basal regulation and sexually dimorphic regulation and induction by the potent CAR activator TCPOBOP and the moderate CAR activator Nonylphenol (NP). NP is an environmental estrogen and one of the most commonly found environmental toxicants in Europe and the United States. Previous studies have demonstrated that NP induces several CYPs in a sexually dimorphic manner, however the role of CAR in regulating NP-mediated sexually dimorphic P450 expression and induction has not been elucidated. Therefore, wild-type and CAR-null male and female mice were treated with honey as a carrier, NP, or TCPOBOP and CYP expression monitored by QPCR and Western blotting. CAR basally regulates the expression of Cyp2c29, Cyp2b13, and potentially Cyp2b10 as demonstrated by QPCR. Furthermore, we observed a shift in the testosterone 6alpha/15alpha-hydroxylase ratio in untreated CAR-null female mice to the male pattern, which indicates an alteration in androgen status and suggests a role for androgens as CAR inverse agonists. Xenobiotic-treatments with NP and TCPOBOP induced Cyp2b10, Cyp2c29, and Cyp3a11 in a CAR-mediated fashion; however NP only induced these CYPs in females and TCPOBOP induced these CYPs in both males and females. Interestingly, Cyp2a4, was only induced in wild-type male mice by TCPOBOP suggesting Cyp2a4 induction is not sensitive to CAR-mediated induction in females. Overall, TCPOBOP and NP show similar CYP induction profiles in females, but widely different profiles in males potentially related to lower sensitivity of males to either indirect or moderate CAR activators such as NP. In summary, CAR regulates the basal and chemically inducible expression of several sexually dimorphic xenobiotic metabolizing P450s in a manner that varies depending on the ligand.

The environmental estrogen, nonylphenol, activates the constitutive androstane receptor.

Nonylphenol (NP) and its parent compounds, the nonylphenol ethoxylates are some of the most prevalent chemicals found in U.S. waterways. NP is also resistant to biodegradation and is a known environmental estrogen, which makes NP a chemical of concern. Our data show that NP also activates the constitutive androstane receptor (CAR), an orphan nuclear receptor important in the induction of detoxification enzymes, including the P450s. Transactivation assays demonstrate that NP increases murine CAR (mCAR) transcriptional activity, and NP treatment can overcome the inhibitory effects of the inverse agonist, androstanol, on mCAR activation. Treatment of wild-type (CAR +/+) mice with NP at 50 or 75 mg/kg/day increases Cyp2b protein expression in a dose-dependent manner as demonstrated by Western blotting, and was confirmed by quantitative reverse transcription-PCR of Cyp2b10 transcript levels. CAR-null (CAR -/-) mice show no increased expression of Cyp2b following NP treatment, indicating that CAR is required for NP-mediated Cyp2b induction. In addition, NP increases the translocation of CAR into the nucleus, which is the key step in the commencement of CAR's transcriptional activity. NP also induced CYP2B6 in primary human hepatocytes, and increased Cyp2b10 messenger RNA and protein expression in humanized CAR mice, indicating that NP is an activator of human CAR as well. In conclusion, NP is a CAR activator, and this was demonstrated in vitro with transactivation assays and in vivo with transgenic CAR mouse models.

Modulation of recombinant small-conductance Ca(2+)-activated K(+) channels by the muscle relaxant chlorzoxazone and structurally related compounds.

Using the patch clamp technique we investigated the effects of the centrally acting muscle relaxant chlorzoxazone and three structurally related compounds, 1-ethyl-2-benzimidazolinone (1-EBIO), zoxazolamine, and 1,3-dihydro-1-[2-hydroxy-5-(triflu oromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS 1619) on recombinant rat brain SK2 channels (rSK2 channels) expressed in HEK293 mammalian cells. SK channels are small conductance K(+) channels normally activated by a rise in intracellular Ca(2+) concentration; they modulate the electrical excitability in neurons and neuroendocrine cells. When applied externally, chlorzoxazone, 1-EBIO, and zoxazolamine activated rSK2 channel currents in cells dialyzed with a nominally Ca(2+)-free intracellular solution. The activation was reversible, reproducible, and depended on the chemical structure and concentration. The order of potency was 1-EBIO > chlorzoxazone > zoxazolamine. Activation of rSK2 channels by chlorzoxazone, 1-EBIO, and zoxazolamine declined at higher drug concentrations. Zoxazolamine, when applied in combination with chlorzoxazone or 1-EBIO, partially inhibited the rSK2 channel current responses, suggesting a partial-agonist mode of action. 1-EBIO failed to activate rSK2 channel currents when applied to excised inside-out membrane patches exposed to a Ca(2+)-free intracellular solution. In contrast, 1-EBIO activated rSK2 currents in a concentration-dependent manner when coapplied to the patches with a solution containing 20 nM free Ca(2+). NS 1619 did not activate rSK2 channel currents; it inhibited rSK2 channel currents activated by the other three test compounds or by high intracellular Ca(2+). We conclude that chlorzoxazone and its derivatives act through a common mechanism to modulate rSK2 channels, and SK channel modulation in the brain may partly underlie the clinical effects of chlorzoxazone.

Pharmacological activation of cloned intermediate- and small-conductance Ca(2+)-activated K(+) channels.

We previously characterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as the clinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine (ZOX), as pharmacological activators of the intermediate-conductance Ca(2+)-activated K(+) channel, hIK1. The mechanism of activation of hIK1, as well as the highly homologous small-conductance, Ca(2+)-dependent K(+) channel, rSK2, was determined following heterologous expression in Xenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced a concentration-dependent activation of hIK1, with half-maximal (K(1/2)) values of 84 microM and 98 microM, respectively. Similarly, CZ activated rSK2 with a K(1/2) of 87 microM. In the absence of CZ, the Ca(2+)-dependent activation of hIK1 was best fit with a K(1/2) of 700 nM and a Hill coefficient (n) of 2.0. rSK2 was activated by Ca(2+) with a K(1/2) of 700 nM and an n of 2.5. Addition of CZ had no effect on either the K(1/2) or n for Ca(2+)-dependent activation of either hIK1 or rSK2. Rather, CZ increased channel activity at all Ca(2+) concentrations (V(max)). Event-duration analysis revealed hIK1 was minimally described by two open and three closed times. Activation by 1-EBIO had no effect on tau(o1), tau(o2), or tau(c1), whereas tau(c2) and tau(c3) were reduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. In conclusion, we define 1-EBIO, CZ, and ZOX as the first known activators of hIK1 and rSK2. Openers of IK and SK channels may be therapeutically beneficial in cystic fibrosis and vascular diseases.

Stimulation of Cl(-) secretion by chlorzoxazone.

We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K(+) channels (K(Ca)), thereby stimulating Cl(-) secretion across several epithelia. In our pursuit to identify potent modulators of Cl(-) secretion that may be useful to overcome the Cl(-) secretory defect in cystic fibrosis (CF), we have identified chlorzoxazone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting muscle relaxant, as a stimulator of Cl(-) secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl(-) secretory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K(Ca). In nystatin-permeabilized monolayers, chlorzoxazone stimulated a basolateral membrane I(K), which was inhibited by CTX and also stimulated an apical I(Cl) that was inhibited by glibenclamide, indicating that the G(Cl) responsible for this I(Cl) may be cystic fibrosis transmembrane conductance regulator (CFTR). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated (86)Rb(+) uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifying K(+) channel, which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate K(Ca), whereas zoxazolamine (2-amino-5-chlorzoxazole) showed a similar response profile as chlorzoxazone. In normal human nasal epithelium, chlorzoxazone elicited hyperpolarization of the potential difference that was similar in magnitude to isoproterenol. However, in the nasal epithelium of CF patients with the DeltaF508 mutation of CFTR, there was no detectable Cl(-) secretory response to chlorzoxazone. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl(-) secretion in normal airway epithelium in vitro and in vivo, and suggest that stimulation requires functional CFTR in the epithelia.

Acute and subchronic evaluation of Indigofera arrecta: absence of both toxicity and modulation of selected cytochrome P450 isozymes in ddY mice.

Indigofera arrecta, an anti diabetic plant was investigated in ddY mice to determine its acute and subchronic effects, and whether it modulated hepatic cytochrome P450 (CYP) isozymes and glutathione (GSH). No mortality was observed in the acute (up to 10 g I. arrecta/kg body wt, p.o.) and subchronic (2 g I. arrecta/kg body wt, p.o. daily for 30 days) studies. The extract did not alter haematological indices, serum and tissue lipids and glutathione but lowered serum bile acids. The latter phenomenon is under further investigation. Neither the duration of pentobarbital (PB) and zoxazolamine (ZA) effects in vivo, nor CYP-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-depentylase (PROD) and p-nitrophenol hydroxylase (PNPH) activities in vitro were altered by I. arrecta. The extract was thus devoid of overt acute and subchronic toxic effects, and did not affect CYPs and GSH whose modulation may cause interactions of components in a multiple drug therapy.

The effect of Rhazya stricta Decne, a traditional medicinal plant, on spontaneous and drug-induced alterations in activity of rats.

The effect of acute and chronic treatment of rats with a lyophilized extract of the leaves of the medicinal plant Rhazya stricta on total and ambulatory activity was studied. Given acutely at single oral doses of 1, 2, 4, and 8 g/kg, the extract produced dose-dependent decreases in total activity and ambulatory activity. Diazepam (20 mg/kg, orally) produced a decrease in rat activity comparable to that produced by a dose of 1 g/kg of the extract. When given daily at an oral dose of 2 g/kg for 21 consecutive days, the extract produced, on the last day of treatment, significant decrease in activity amounting to about 30% of control activity levels. Subcutaneous (SC) treatment of rats with caffeine (7.5, 15, and 30 mg/kg), dose-dependently and significantly increased total activity and ambulatory activity. These effects were dose-dependently attenuated when the extract was given concomitantly with caffeine at oral doses of 1, 2, and 4 mg/kg. Treatment of rats with zoxazolamine alone (10, 20, or 40 mg/kg, SC) or R. Stricta (1 and 4 g/kg orally) alone significantly decreased total and ambulatory activities. Concomitant treatment with zoxazolamine and R. Stricta decreased the rats activity to a greater degree than with either treatment given alone.

Subchronic toxicity studies of sucrose acetate isobutyrate (SAIB) in the rat and dog.

Preliminary short-term toxicity studies of sucrose acetate isobutyrate (SAIB) in the dog demonstrated that addition of this additive to the diet was associated with an increase in liver size and elevated serum alkaline phosphatase activity with no evidence of pathological change by light microscopy. To determine the basis for these changes, a 12-week oral toxicity study of SAIB was conducted in the dog and a similar study was performed in the rat. SAIB was fed in the diet to groups of six beagle dogs of each sex at 0, 0.5, 1.0, 2.0 and 4.0%. SAIB was also fed to groups of 40 Sprague-Dawley rats of each sex at levels of 0, 2.5, 5.0 and 10.0%. In the rat study, in addition to routine toxicology parameters, hepatic microsomal enzyme induction was determined using a zoxazolamine hypnotic test, urinary ascorbic acid excretion and determination of hepatic carboxylesterase activity. Sodium phenobarbital was fed to groups of 20 rats of each sex at a dose of 100 mg/kg body weight/day by gavage as a positive control for hepatic microsomal enzyme induction. In the dog study, routine toxicological tests were supplemented by tests for bromsulfophthalein (BSP) retention, histochemical staining of liver sections for glycogen, phosphorylase, succinate dehydrogenase, and acid and alkaline phosphatases. Levels of liver lipid, protein, glycogen and carboxylesterase activity were also determined. Electron microscopic examinations were made on liver sections from the dog study at the end of the 12-week SAIB feeding period and after a 2-week withdrawal period. Administration of SAIB to rats did not reveal evidence of any effect on hepatobiliary function, and there was no indication of microsomal enzyme induction. Body weight gain of male rats fed SAIB was decreased, probably as the result of decreased palatability of the diet; SAIB did not affect body weight gain in females. The changes observed in the dogs fed SAIB included increased serum alkaline phosphatase activity with no change in serum alanine aminotransferase, aspartate aminotransferase or lactic dehydrogenase activity and no change in serum electrolyte, serum protein, glucose or bilirubin levels. No haematological changes were observed. BSP retention was observed at all SAIB dose levels. There were no SAIB-related pathological changes in any organ when examined by light microscopy. Examination by electron microscope revealed dilatation of bile canaliculi and an increase in smooth endoplasmic reticulum compared with controls. Histochemical studies also indicated increased enzyme activity of the bile canaliculi. The electron-microscope-revealed changes were completely reversed during a 2-week treatment withdrawal period. The dog study did not establish a no-effect level for changes in hepatobiliary function induced by feeding SAIB.

Zoxazolamine-induced paralysis in two rat substrains: differences in hepatic drug metabolism.

Aldehyde dehydrogenase (ALDH) is involved in the metabolism of endogenous and exogenous aldehydes originating from biogenic amines, lipids, food and drugs. Rat liver contains at least two cytosolic ALDHs that can be stimulated by inducers of drug metabolism. Phenobarbital- type inducers increase ALDH1 activity while polycyclic aromatic hydrocarbons (such as benzo[alpha]pyrene) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increase ALDH3c isoenzyme activity. Two rat substrains were isolated according to a different induction of hepatic ALDH after treatment with phenobarbital (PB). Animals that responded to treatment (RR) and those that did not respond (rr) were inbred and divided into two homogenous groups. These animals constituted an ideal experimental model due to their common origin. Apart from the dramatic induction of cytosolic ALDH1 and ALDH3c, the effects of PB on pentoxy-, ethoxy- and methoxy-resorufin-O-dealkylase (P-, E-, and MROD) between the two substrains were also studied. 3-Methylcholanthrene (3MC) greatly increased ALDH3c levels in both substrains, although it was slightly more pronounced in the rr rats, in which it was assessed either as ALDH3c or as total cytosolic ALDH. A similar trend was also noted in EROD, PROD and MROD activities. Dealkylation of the methoxy group was found to be statistically different between the two substrains (rr > RR). The relevance of the biochemical findings with the in vivo hepatic capacity for drug metabolism was investigated by measuring the duration of zoxazolamine paralysis. Both animal substrains were tested with zoxazolamine either without pretreatment or after administration of PB or 3MC: the paralysis produced by zoxazolamine lasted for a longer period in rr than in RR rats. After pretreatment with PB, the duration of paralysis was greatly reduced, but the differences between the two substrains remained. Pretreatment with various doses of 3MC produced differences in the duration of paralysis in RR and rr rats, although the time period was much shorter than that observed in control animals.

Effect of 2,4-disubstituted thiazol-5yl-aminoketones on inflammation. In vitro and in vivo biological studies: a structure-activity approach.

Some modified novel thiazol-5yl-aminoketones were evaluated for their anti-inflammatory, analgesic and antiproteolytic activities. Their inhibitory activity on 12-lipoxygenase (12-LO) and beta-glucuronidase in vitro was estimated. Their interaction with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and their RM values were also determined. For two of them, the effect on zoxazolamine-induced paralysis after a prolonged treatment was determined. The duration of paralysis for the same compounds, (only one administration, one before zoxazolamine injection) was recorded too. The 2-amino substituted derivatives seem to be more potent in comparison with the 2-phenyl. The tested compounds were found to influence proteolysis but not the activities at beta-glucuronidase and 12-LO. Their interaction with DPPH was mild. Compound 2 seems to modify the activity of the hepatic drug metabolizing enzymes. In conclusion, their activity is related to certain structural characteristics.

In vivo modulation of the rat cytochrome P450 1A1 by double-stranded phosphorothioate oligodeoxynucleotides.

CYP1A1 gene expression is regulated by known cis- and transacting elements controlling inhibition and induction of CYP1A1 transcription. The influence of a double-stranded phosphorothioate oligonucleotide (dsODN) with sequence identical to the CYP1A1 negative regulatory element (NRE) was examined in Sprague-Dawley rats. Two strategies were employed: (i) two single-stranded complementary 25-mer ODNs that form a double-stranded ODN (ODN1) and (ii) a 54-base, self-complementary ODN which forms a dsODN hairpin (ODN2). A dsODN hairpin with scrambled NRE sequence was evaluated as a control (ODN3). Zoxazolamine paralysis times, an in vivo marker of CYP1A1 activity, were reduced from 184 +/- 18 min in saline-treated rats to 103 +/- 12.5 min 24 hr after a single 1.7-mg ODN1 iv injection. Liver microsomal EROD, an in vitro marker of CYP1A1/2 activity, was increased from 210 +/- 10 pmol in saline-treated animals to 703 +/- 73 and 623 +/- 89 pmol resorufin/mg protein/min after iv ODN1 and iv ODN2, respectively. ODN1's activity did not change PNP hydroxylation and PROD, markers of CYPs 2E1 and 2B1/2. ODN2 did not significantly change PNP but did significantly alter PROD. The ODN3 did not cause any significant changes in any assay measured. The ODN1-induced responses in ZX paralysis and EROD were observed post-iv injection, but not following ip injection of ODN1. Western blot analysis of ODN1- and HPO-treated rat liver microsomes also revealed increased in CYP1A1 protein. These data indicate double-stranded ODNs mimic the cis-acting NRE in vivo inducing CYP1A1 in the absence of other xenobiotics.

Intensity of Schistosoma mansoni infection determines alterations in hepatic drug metabolism.

Zoxazolamine paralysis times have been used as a probe to measure the activities of cytochrome P450 1A1 in mice in vivo. The results indicate that male and female mice of the BALB/c and CBA/J strain do not show altered paralysis times if infected with less than 4 worm pairs. Alterations were observed only in animals harbouring more than 5 worm pairs irrespective of the sex or strain of mouse being used. The study has extended the findings of other workers showing that mice infected with fewer than 4-5 worm pairs of S. mansoni do not show any alteration in the metabolism of pentobarbital in vivo.

Effect of spironolactone on dimethyl mercury toxicity. A possible molecular mechanism.

The protective activity of spironolactone (CAS 52-01-7) against dimethyl mercury intoxication was studied. Dimethyl mercury increased serum glutamate pyruvate transaminase (SGPT), serum bilirubin, blood urea nitrogen (BUN), and caused impairment of the drug metabolic activity of rat liver in vivo and in vitro. It also caused a severe neuropathy to these animals. Administration of spironolactone caused a reduction of dimethyl mercury toxicity. It decreased the values of SGPT, bilirubin and BUN, and restored the impaired drug metabolism caused by dimethyl mercury. The neuropathy produced after administration of dimethyl mercury was only mildly ameliorated by the treatment with spironolactone. Pregnenolone-16 alpha-carbonitrile (PCN), a potent microsomal enzyme inducer, had only a weak action, with the expected exception of the repair of the impaired drug metabolism of the liver. A mechanism of the protective action of spironolactone against dimethyl mercury intoxication is proposed. It is suggested that both the ability to induce drug metabolizing enzymes, here demethylases, and the capacity to bind to the demethylated metabolite of the organic mercurial, giving a non toxic, easily excretable complex should coexist in the protective molecule.

Effects of furazolidone on duration of righting reflex loss induced with hexobarbital and zoxazolamine in the rat.

Effects of furazolidone (FZ) on the sleeping time induced with hexobarbital (HEX) and paralysis time induced by zoxazolamine (ZOX) were investigated by measuring the length of time required to recover from righting reflex loss in rats after oral administration of FZ at doses of 50, 100, 200 and 400 mg/kg/day for 4 successive days. Administration of 50 mg/kg to rats of both sexes induced no effect on the HEX sleeping time, but of 100 mg/kg FZ or more induced prolongation of sleeping time dose-dependently. In female rats, HEX sleeping time of the control group was twice that of the male rats, but HEX sleeping time after receiving FZ above 200 mg/kg was approximately the same as in the male rats. ZOX paralysis time exhibited no sex differences in the control rats, and it was significantly prolonged by FZ at a dose of 100 mg/kg or more. No significant differences in blood levels of HEX and ZOX at the time of recovery were found between the control and FZ treated rats, suggesting that FZ produced prolongation of the drug effects was due to the maintenance of the blood levels rather than the change in the sensitivities of rats at the receptor sites. Body weight gains were inhibited in the rats treated with FZ at doses over 100 mg/kg. Cytochrome P-450 content in hepatic microsomes in the rats which received 100 mg/kg FZ were slightly increased. It is suggested that successive oral administration of FZ to rats at high doses impaired drug clearance and this resulted in the prolongation of HEX sleeping and ZOX paralysis times.

Effects of ciprofloxacin and enrofloxacin on zoxazolamine kinetics, plasma concentration and sleeping times in mice.

The treatment of CD1 male mice with either ciprofloxacin (CP) or enrofloxacin (EF) prior to zoxazolamine (ZX) administration increased the mean ZX sleeping times to, respectively, 162 and 156% of the control (ZX alone). At the end of the sleeping time, the mean ZX plasma concentration in controls was 27.2 micrograms/ml and was not different in EF- or CP-treated groups (87% and 95% of controls, respectively). The animals coadministered with CP or EF and ZX eliminated the latter more slowly than the controls. The estimated zero-time drug concentration of the disposition curves of both the CP- and EF-treated groups as well as the apparent half-life of elimination and apparent overall rate of elimination of the CP-treated group were different from the control values.

On central muscle relaxants, strychnine-insensitive glycine receptors and two old drugs: zoxazolamine and HA-966.

Zoxazolamine is in the centrally-acting muscle relaxant class of drugs, which reportedly act by decreasing CNS interneuronal activity. These drugs, but not anxiolytics, decrease dopaminergic turnover and induce a pacemaker-like discharge pattern in dopaminergic neurons. A mechanism for these effects was not found in previous reports. We observed that (+)-HA-966, an inhibitor of the glycine modulatory site on the NMDA receptor, has a similar effect on dopaminergic impulse flow, which suggested that this may be the possible site of action of classical muscle relaxants. However, a competitive antagonist of NMDA receptors, NPC-12626, had little effect on impulse flow. Binding of 20 nM [3H]-glycine to cortical synaptosomal membranes was inhibited by (+)-HA-966, IC50 = 3.16 microM, but only poorly by zoxazolamine, IC50 V 474 microM, and chlorzoxazone, a related drug, caused no displacement. The drugs were then tested for protection from amphetamine neurotoxicity. Neither 50 mg/kg zoxazolamine nor 30 mg/kg (+)-HA-966 prevented (+)-amphetamine (0.1 mmol/kg plus 10 mg/kg iprindole) depletion of striatal dopamine (DA), but 3.0 mg/kg of MK-801, a non-competitive NMDA receptor antagonist, did protect DA content. Since baclofen induces a regular firing rate in DA neurons, zoxazolamine and (+)-HA-966 were tested for displacement of 10 nM [3H]-1-baclofen from cortical synaptosomal GABAb receptors, but were ineffective. Thus, the effects of these muscle relaxants on DA neurons are mediated by a mechanism other than strychnine-insensitive glycine or GABAb receptors.