Effect of morphinone on opiate receptor binding and morphine-elicited analgesia

Specific binding of ³H-naloxone to opiate receptors was found to be irreversibly inactivated by morphinone. This inactivation exhibited pseudo-first-order kinetics. The presence of sulfhydryl compounds or morphine during incubation with morphinone proved good protection. Morphinone-pretreated mice blocked the analgesic effect of morphine. The possible mechanism for these observations is proposed as foolows: morphinone binds covalently to sulfhydryl group of opiate receptors, and inactivates irreversibly opiate binding sites, thus blocking the analgesic effect of morphine.     

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. $\text{In vitro}$ CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.     

Protective effect of reduced glutathione against cisplatin-induced renal and systemic toxicity and its influence on the therapeutic activity of the antitumor drug

Reduced glutathione has been shown to be an effective protector against cisplatin-induced nephrotoxicity of potential clinical value, since it does not reduce antitumor activity of the cytotoxic drug. This paper extends previous observations on the protective potential of reduced glutathione against cisplatin-induced nephrotoxicity, in different rodent models. Following i.v. administration, glutathione protection against cisplatin-induced nephrotoxicity was found to be critically dependent on timing of thiol administration. Whereas the sulfhydryl compound provided almost complete protection in CD rats, the protective effect against toxic renal damage was only partial in mice of different strains. In spite of the modest protection against kidney toxicity, glutathione reduced lethal toxicity in the mouse. Under the same experimental conditions at protective dose levels, the tripeptide thiol did not interfere with the antitumor effectiveness of cisplatin, in any of the tumor models examined. The kidney content of non-protein sulfhydryls of CD rats produced by the effective dose of glutathione was markedly higher than that found in the mouse treated with the same dose. This finding is consistent with a differential protection provided by glutathione against cisplatin-induced renal toxicity in these species.     

Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen

Acetaminophen, a widely prescribed analgesic that causes fulminant hepatic necrosis in overdosed humans, produced varying degrees of hepatotoxixity in mice, rats, hamsters, guinea pigs and rabbits. The severity of hepatic injury paralleled the rate of activation of acetaminophen by hepatic microsomal enzymes to a potent arylating agent. The severity of hepatic damage in various species also correlated directly with the rate of hepatic glutathione depletion after acetaminophen. These findings support the hypothesis that the electrophilic arylating agent formed from acetaminophen $\text{in}$$\text{vibo}$ is preferentially detoxified by conjugation with glutathione and that arylation of hepatic macromolecules occurs only when glutathione availability is exceeded. Since N-hydroxylation of another N-acetylarylamine (2-acetylaminofluorene) occurs to a much greater extent in the species that are susceptible to acetaminophen-induced hepatic necrosis, the data also are consistent with the hypothesis that the toxic metabolite of acetaminophen results from N-hydroxylation.     

Effects on drug metabolism of carbaryl and 1-naphthol in the mouse

The effects of carbyl and 1-naphthol on hepatic microsomal drug-metabolizing enzyme systems were investigated. The agents were fed at a level of 25 mmol/kg of feed to groups of young male Swiss-Webster mice for 14-day periods. Body weight was depressed by carbaryl, but not by 1-naphthol. The rates of $\text{in vitro}$ metabolism of aniline and benzphetamine were greater than control rates in livers of mice fed carbaryl, but the rate of $\text{in vivo}$ hydrolysis of the carbamate insecticide Zectran was decreased by carbaryl feeding. Administration of 1-naphthol did not change the rates of $\text{in vitro}$ metabolism of either aniline or benzphetamine. Hepatic microsomal concentrations of cytochromes P-450 and b₅ were increased by carbaryl, but feeding of 1-naphthol did not affect levels of either cytochrome. Radiolabeled pentobarbital disappeared from the blood of carbaryl-fed mice more rapidly than from the blood of control animals, and carbaryl-fed mice slept a shorter period of time than controls following pentobarbital administration. The LD50 of an acute oral dose of carbaryl was increased two-fold by feeding carbaryl for 14 days. It was concluded that carbaryl is a weak inducer of hepatic microsomal drug-metabolizing activity, and that the effects observed are not likely due to 1-naphthol.     

Mechanism of development of tolerance to injected morphine by guinea pig ileum.

Injection of a large dose of morphine into a guinea pig results in a block of electrically-induced contractions of the ileum $\text{in vitro}$. A similar dose is almost ineffective in guinea pigs given morphine chronically. The time course for development of this tolerance has been determined in guinea pigs injected twice daily with morphine 100 mg/kg and challenged on various days with 750 mg/kg of the drug. Animals similarly injected but not challenged served as controls. The inhibitory effect of the challenging dose on electrical stimulation of longitudinal muscle decreased with successive days of morphine administration; by the 10th day there was almost complete tolerance to the challenging dose. Sensitivity of the tissues of chronically morphinized unchallenged controls towards acetylcholine, serotonin, histamine and norepinephrine was essentially the same as that of naive animals. The potency of morphine $\text{in vitro}$ in blocking electrical stimulation was also unchanged by chronic morphine administration in the above manner. Thus tolerance to injected morphine cannot be explained by reduced affinity of the drug for the opiate receptor. Tissues of chronically morphinized animals gave a contracture with naloxone, the extent of the contracture increasing with time of drug administration. This naloxone effect is attributed to displacement of morphine from a new opiate receptor site induced during morphine administration. It is suggested that this new receptor is involved in tolerance to injected morphine as well as some aspects of the withdrawal syndrome.     

Denaturation of cytochrome P-450 by cyclophosphamide metabolites

Cyclophosphamide (CP) metabolites, acrolein and 4-hydroxy-CP, were found to denature rat liver microsomal cytochrome P-450, whereas another metabolite, phosphoramide mustard, CP $\text{per}\text{se}$ or its analog Ifosfamide had no effect. The denaturation produced by CP metabolites could be blocked by cysteine, suggesting an interaction between CP metabolite(s) and sulfhydryl groups in cysteine and probably in cytochrome P-450. These studies might explain the biochemical basis of the specific depression of various microsomal mixed function oxygenase activities produced by high doses of CP.     

Acrylonitrile and tissue glutathione: Differential effect of acute and chronic interactions

Acrylonitrile (vinyl cyanide) is a reactive chemical extensively used in the synthesis of buna rubber and polymerized plastics (e.g., disposable bottles). A single dose of the chemical causes adrenal hemorrhage, while chronic ingesting results in adrenocortical hypofunction. Present acute experiments in rats show a rapid time- and dose-dependent $\text{decrease}$ in reduced glutathione (GSH) in the liver, lung, kidney and adrenal. The diminution of cerebral GSH concentration is gradual and seems to correlate with the occurrence of mortality in acute experiments. Chronic ingestion of acrylonitrile in drinking water results in a dose dependent $\text{increase}$ of hepatic GSH concentration, similar to that caused by chemical carcinogens.     

Acute inhibition of microsomal drug metabolism by propoxyphene in narcotic tolerant/dependent mice

Propoxyphene (Darvon) was compared to SKF 525-A, a prototypical inhibitor of hepatic microsomal mixed function oxidases, to assess propoxyphene's potential to inhibit drug metabolism in morphine tolerant/dependent mice. $\text{In vitro}$, both propoxyphene (K_i = 3.5 × 10^?5M) and SKF 525-A (K_i = 4.3 × 10^?6M) inhibited the activity of aminopyrine N-demethylase competitively in hepatic microsomes from tolerant/dependent animals. Propoxyphene and SKF 525-A were weaker, noncompetitive inhibitors of aniline hydroxylase activity. $\text{In vivo}$, equimolar doses (0.24 mmoles/kg, i.p.) of each compound inhibited both of the above monooxygenases in the 10,000$\text{g}$ supernatant fractions of livers from the tolerant/ dependent animals. Propoxyphene was 40–50% as potent an inhibitor of these activities as SKF 525-A. A dose (300 mg/kg) of propoxyphene napsylate, shown to prevent narcotic abstinence signs with no observable toxicity in withdrawing mice, significantly prolonged the blood levels of injected pentobarbital and tripled pentobarbital sleeping time in these animals. When administered at 300 mg/kg chronically, propoxyphene napsylate acted as an inducer of its own metabolism. Propoxyphene napsylate, then, given acutely to narcotic tolerant/dependent mice, is a potent inhibitor of microsomal drug metabolizing capacity. Given chronically, it enhances this capability.     

Effects of yohimbine and naltrexone in counteraction of the morphine straub tail and the amphetamine-potentiated morphine straub tail in mice

The alpha-adrenergic blocking agent, yohimbine, prevented the production of the morphine Straub tail reaction in mice, although on a mg dose basis it was only about $\text{1}\text{400}$ as potent as the narcotic antagonist naltrexone by subcutaneous injection. Likewise, yohimbine prevented the potentiation of the morphine Straub tail reaction by amphetamine, being about $\text{1}\text{170}$ as active as naltrexone. Preliminary studies with another alpha-adrenergic blocking agent, phentolamine, indicated that it also inhibited the production of the Straub tail reaction by morphine, although it appeared to be somewhat weaker than yohimbine in this respect. These results suggest the involvement of alpha-adrenergic mechanisms in the production of the morphine Straub tail reaction and in the potentiation of the morphine Straub tail reaction by amphetamine.     

Dopamine-sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine.

The addition of narcotic analgesics $\text{in vitro}$ to nerve ending preparations from rat caudate nucleus in an assay of adenylate cyclase activity (AC) resulted in an inhibition of basal AC only at drug concentrations of 10−⁴M or higher, and no inhibition of dopamine-stimulated (DA) AC at these drug concentrations. The acute administration of morphine at a moderately high dose (60 mg/kg) produced an increase in striatal cAMP levels, and increases in basal and DA-AC in caudate nerve-endings. In morphine-tolerant rats, striatal cAMP levels and basal AC were similar to control values, while DA-AC was elevated. These results suggest: (1) that opiates do not act directly on DA-AC, the ‘dopamine receptor’, and (2) that the observed behavioural DA sensitivity in tolerant animals may be produced by the DA-AC supersensitivity.     

Evidence of possible opiate dependence during the behavioral depressant action of a single dose of morphine

Rats were trained to lever press for food pellets under a 20 response fixed ratio (FR 20) schedule of reinforcement. A single injection of 15 mg morphine SO₄/kg suppressed operant behavior for $\text{1}\text{1}\text{2}\text{–3}\text{1}\text{2}\text{hrs}$, after which time responding resumed at a reduced rate. When 0.25 mg naloxone HCl/kg was given during the recovery phase, the behavioral depressant effect of the narcotic was immediately reversed and operant performance returned to predrug rates. In contrast, when 0.5 mg naloxone/kg was given at this time, operant behavior was abolished for at least 1 hr. Naloxone, at these doses, did not affect responding in drug-naive subjects. These results suggest that a single, relatively low dose of morphine can induce transient dependence which is detectable for several hrs after drug administration, at a time when the acute pharmacological actions of morphine are still apparent.     

The inhibition of morphine: UDP-glucuronyltransferase in rabbit liver microsomes by cyproheptadine.

The reactivity of the non-narcotic substances, cyproheptadine and N-desmethylcyproheptadine with morphine: UDP-glucuronyltransferase was studied in rabbit hepatic microsomal preparations. Cyproheptadine produced a potent competitive inhibition of morphine glucuronidation $\text{in vitro}$ (K_i=0.08 mM) whereas its N-desmethyl derivative was significantly less effective (K_i=0.4 mM). No cyproheptadine glucuronide was formed in these reactions suggesting that cyproheptadine acts as a dead-end inhibitor. Results indicate that the mechanism of the inhibition of morphine: UDP-glucuronyltransferase by cyproheptadine is similar to that produced by opioids and is related to the presence of the N-alkyl group in it structure.     

Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on $\text{in}$$\text{vitro}$ lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it had no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

Lipid metabolism and in vitro production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

The effects of Cloprostenol administration on porcine luteal lipid and arachidonic acid accumulation were examined in relation to luteal $\text{in vitro}$ progesterone and prostaglandin F synthesis in 18 mature gilts at day 12 of the estrous cycle. Basal and net $\text{in vitro}$ release of progesterone from luteal tissue was depressed at 8 hr after treatment whereas net $\text{in vitro}$ release of prostaglandin F was elevated at 8 hr. Inclusion of copper dithiothreitol or reduced glutathione in the incubation media resulted in minor alterations of $\text{in vitro}$ release of progesterone and prostaglandin F and no changes in composition of luteal lipids or fatty acids. Luteal contents of triglyceride had increased by 8 hr after treatment whereas contents of free and esterified cholesterols had increased by 32 hr after Cloprostenol administration. Luteal contents of phospholipid and free fatty acids were not affected by Cloprostenol administration. At 32 hr after treatment, percentages and content of arachidonic acid had increased in luteal cholesterol esters and triglycerides. Although arachidonic acid percentages increased in luteal free fatty acids and phospholipids, calculated arachidonic acid contents did not change following Cloprostenol administration. Induced luteal regression was associated with decreased $\text{in vitro}$ progesterone release, increased $\text{in vitro}$ prostaglandin F release, and accelerated lipid and arachidonic acid accumulation within the corpus luteum. The effects of altered lipid metabolism on release of prostaglandin F could not be defined. However, availability of arachidonic acid did not appear to be rate-limiting in relation to luteal $\text{in vitro}$ prostaglandin F synthesis.     

Stimulation of intralysosomal proteolysis by cysteinyl-glycine,a product of the action of γ-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversible inhibitor of γ-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with $\text{l}\text{-(αS,5S)-α-}\text{amino}\text{-3-}\text{chloro}\text{-4,5-}\text{dihydro}\text{-5-}\text{isoxazoleacetic}$ acid (acivicin), an irreversible inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepated kidney membranes or purified γ-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of γ-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or γ-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of γ-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.     

Interactions of thyrotropin releasing hormone and morphine sulfate in rodents.

Thyrotopin releasing hormone (TRH) produces “wet dog shakes” in rats similar to those observed during morphine withdrawal. The shaking behavior precipitated by morphine abstinence can be exacerbated by TRH administration while the other components of the morphine withdrawal syndrome remain unchanged. Morphine, chlorpromazine, apomorphine, and Δ⁹-tetrahydrocannabinol effectively block shakes induced by either TRH administration or morphine withdrawal. These results suggest the possibility that endogenous TRH may be associated with the “wet dog shakes” observed as a portion of morphine's abstinence syndrome in rats. However, TRH is unable to alter the stereospecific binding of morphine $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$, and naloxone fails to potentiate the number of TRH-induced shakes. TRH has no antinociceptive properties, and it cannot alter those of morphine. These data suggest that more than one neuromechanism may be responsible for shaking behavior in rats.     

p-Aminophenol-induced hepatotoxicity in hamsters: role of glutathione

p-Aminophenol (PAP) is a widely used industrial chemical and a known nephrotoxin. Recently, it was found to also cause hepatotoxicity and glutathione (GSH) depletion in mice. The exact mechanism of liver toxicity is not known. The aims of this study were to determine whether PAP can cause acute hepatotoxicity in hamsters and to further investigate the role of GSH in PAP-induced toxicity. PAP was administered ip to hamsters in doses of 200-800 mg/kg. Liver damage at 24 h after PAP administration was assessed by elevations in plasma enzyme activities and histopathologic examination. GSH and cysteine (Cys) levels in liver at 4 h were determined by HPLC. PAP decreased hepatic GSH concentration to 8% and Cys to 30% of vehicle control values. It increased plasma glutamic pyruvic transaminase (GPT) activity by 47-fold and sorbitol dehydrogenase (SDH) activity by 113-fold. PAP also caused severe centrilobular hepatocellular necrosis. 2(RS)-n-Propylthiazolidine-4(R)-carboxylic acid (PTCA), a Cys precursor, attenuated the PAP-induced decreases in hepatic sulfhydryl levels; GSH and Cys were 39% and 78% of vehicle controls, respectively. PTCA also attenuated the PAP-induced elevations in plasma enzyme activities and hepatic necrosis. It was concluded that PAP hepatotoxicity is associated with depletion of hepatic GSH and can be prevented by PTCA.