Repeated toluene and cyclohexane inhalation produces differential effects on HPA and HPT axes in adolescent male rats.

Misused volatile solvents typically contain toluene (TOL) as the main psychoactive ingredient. Cyclohexane (CHX) can also be present and is considered a safer alternative. Solvent misuse often occurs at early stages of life, leading to permanent neurobehavioral impairment and growth retardation. However, a comprehensive examination of the effects of TOL and CHX on stress regulation and energy balance is lacking. Here, we compared the effect of a binge-pattern exposure to TOL or CHX (4,000 or 8,000 ppm) on body weight, food intake, the hypothalamus-pituitary-adrenal (HPA) and hypothalamus-pituitary-thyroid (HPT) axes in male adolescent Wistar rats. At 8,000 ppm, TOL decreased body weight gain without affecting food intake. In addition, TOL and CHX altered the HPA and HPT axes' function in a solvent- and concentration-dependent manner. The highest TOL concentration produced HPA axis hyperactivation in animals not subjected to stress, which was evidenced by increased corticotropin-releasing-factor (CRF) release from the median eminence (ME), elevated adrenocorticotropin hormone (ACTH) and corticosterone serum levels, and decreased CRF mRNA levels in the hypothalamic paraventricular nucleus (PVN). TOL (8,000 ppm) also increased triiodothyronine (T3) serum levels, decreased pro-thyrotropin-releasing-hormone (pro-TRH) mRNA transcription in the PVN, pro-TRH content in the ME, and serum thyroid stimulating hormone (TSH) levels. CHX did not affect the HPA axis. We propose that the increased HPT axis activity induced by TOL can be related to the impaired body weight gain associated with inhalant misuse. These findings may contribute to a better understanding of the effects of the misused solvents TOL and CHX.

Role of main neuroendocrine pathways activated by swim stress on mast cell-dependent peritoneal TNF production after LPS administration in mice.

OBJECTIVE AND DESIGN: To characterize the effects of swim stress on the early mast cell (MC)-dependent peritoneal production of TNF in response to lipopolysaccharide (LPS) administration in mice, identifying the neuroendocrine mediators involved. SUBJECTS: Ten to twelve-week-old Swiss Webster, C57BL/6 J or c-Kit (Wsh/Wsh) mice were used. TREATMENT: Animals were intraperitoneally challenged with LPS at different times after forced swimming (FS) and peak TNF production was determined in peritoneal washes at optimal time after LPS administration. Selective blockage of main neuroendocrine pathways was performed before swim stress. METHODS: TNF concentrations were determined by ELISA. RESULTS: FS provoked an immediate and transient inhibition of LPS-elicited, MC-dependent TNF accumulation in peritoneum, which lasted around 30 min. Suppresive effects of FS were absent on MC-deficient c-Kit (Wsh/Wsh) mice but were recovered after reconstitution with MC. Adrenalectomy or DSP4 administration increased basal ip TNF levels and enhanced LPS-induced TNF release without any effect on stress-induced inhibitory effects, mifepristone did not produce any change on stress-induced inhibition, whereas mecamylamine administration increased basals and attenuated stress effects. CONCLUSIONS: Swim stress transiently inhibits the canonical MC-dependent response of TNF production in response to LPS in murine peritoneal cavity with the main participation of the cholinergic anti-inflammatory reflex.

Repeated toluene exposure modifies the acetylation pattern of histones H3 and H4 in the rat brain.

Toluene is a volatile organic solvent with addictive potential that exhibits similarities in its physiological effects and modes of action to other addictive drugs. Despite its widespread abuse, the molecular mechanisms driving the response and adaptation of the organism to this drug are not fully understood. In recent years, different epigenetic mechanisms that modulate gene expression have been shown to be associated to cocaine, amphetamine and alcohol misuse-induced alterations in neuronal function. For example, it has been demonstrated that drug consumption induces variations in histone acetylation levels in brain reward regions and these play a relevant role on the abuse-associated behavioral plasticity. In order to decipher whether repeated toluene exposure could mediate epigenetic changes in the rat brain, we here analyzed the acetylation pattern of histones H3 and H4 in three brain areas that have been previously associated to substance abuse reward pathways: the Nucleus Accumbens (NAc), the Ventral Tegmental Area (VTA) and the Central Amygdala (CeA). Using immunofluorescence analysis of brain sections with specific antibodies that recognize the acetylated forms of histones H3 and H4, we demonstrate that chronic toluene inhalation differentially modifies histone H3 and H4 acetylation in the NAc and the VTA while no effect is observed in the CeA. Our results suggest that the activity of chromatin-modifying enzymes such as histone de-acetylases (HDACs) in certain brain areas are responsive to toluene inhalation and might be crucial mediators in the addictive response to toluene.

Toluene and TCE decrease binding to mu-opioid receptors, but not to benzodiazepine and NMDA receptors in mouse brain.

In vitro and in vivo studies have shown that abused solvents affect different neurotransmitter systems, including the GABAergic, glutamatergic, and opioidergic. The first purpose of this study was to determine in mice whether an acute exposure to 4,000 ppm toluene or 12,000 ppm 1,1,1-trichloroethane (TCE) modifies receptor binding levels to: (a) DAMGO, a mu-opioid receptor selective agonist; (b) MK-801, a noncompetitive selective NMDA-receptor antagonist; and (c) flunitrazepam, a benzodiazepine binding site selective agonist. In addition, in separate groups of animals, nociceptive effects of toluene alone or co-administered with morphine were evaluated in the hot-plate test. Mice were exposed to toluene or TCE in static exposure chambers for 30 min, and their brains were removed 24 h later for autoradiography. Acute toluene inhalation produced a significant decrease in mu-opioid receptor binding levels in cingulate and piriform cortices, caudate putamen, thalamus, amygdala, and periaqueductal gray, whereas TCE significantly decreased mu-opioid receptor levels, but only in thalamus and periaqueductal gray. Both toluene and TCE decreased benzodiazepine receptor binding levels in discrete brain areas, but had no effect on NMDA receptor levels. In the hot-plate test, a single toluene exposure counteracted morphine antinociceptive response when the solvent exposure time was immediately followed by morphine treatment, but not when morphine was administered 24, 48, 72, and 96 h later. However, co-administration of morphine and toluene 24, 48, 72, and 96 h after the single solvent exposure resulted in morphine-induced analgesia blockade. Present results suggest that mu-opioid receptors are an important molecular target for organic solvents, and that the inhalation of these compounds may affect the analgesic properties of opioids.

Toluene increases acute thermonociception in mice.

Toluene is an abused solvent widely used in several commercial products. Recent evidence indicates that this solvent is a non-competitive inhibitor of NMDA receptors. Since NMDA receptors have been implicated in pain, this paper describes studies of the effects of increasing concentrations of inhaled toluene on nociception. Swiss Webster mice were exposed to toluene (500-8000 ppm) in static exposure chambers for 30 min. After completing the exposure period, animals were tested for nociception using the hot plate test. Toluene dose-dependently increased nociception as reflected by shorter latencies for the reflex, paw-lick and escape responses in toluene-treated mice with respect to their controls (animals exposed to air). In order to determine the possible role of opioids in this response, morphine (1-10 mg/kg) was injected before toluene inhalation. Toluene was not able to block morphine-induced antinocieption, however, it produced a shift of the morphine dose-response curve to lower effects, suggesting a physiological antagonism. No potentiation was seen when toluene was administered in combination with naloxone. Present results suggest that toluene increases nociception via neurotransmitter systems others than the glutamatergic.

Effects of volatile solvents on recombinant N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

1. We have previously shown that toluene dose-dependently inhibits recombinant N-methyl-D-aspartate (NMDA) receptors at micromolar concentrations. This inhibition was rapid, almost complete and reversible. The NR1/2B combination was the most sensitive receptor subtype tested with an IC(50) value for toluene of 0.17 mM. 2. We now report on the effects of other commonly abused solvents (benzene, m-xylene, ethylbenzene, propylbenzene, 1,1,1-trichlorethane (TCE) and those of a convulsive solvent, 2,2,2-trifluoroethyl ether (flurothyl), on NMDA-induced currents measured in XENOPUS oocytes expressing NR1/2A or NR1/2B receptor subtypes. 3. All of the alkylbenzenes and TCE produced a reversible inhibition of NMDA-induced currents that was dose- and subunit-dependent. The NR1/2B receptor subtype was several times more sensitive to these compounds than the NR1/2A subtype. 4. The convulsant solvent flurothyl had no effect on NMDA responses in oocytes but potently inhibited ion flux through recombinant GABA receptors expressed in oocytes. 5. Overall, these results suggest that abused solvents display pharmacological selectivity and that NR1/2B NMDA receptors may be an important target for the actions of these compounds on the brain.

Anxiolytic-like actions of toluene in the burying behavior and plus-maze tests: differences in sensitivity between 5-HT(1B) knockout and wild-type mice.

This paper compares the anxiolytic-like actions of toluene in two anxiety paradigms, the burying behavior and plus-maze tests, in 5-HT(1B) knockout (KO) and 129/Sv-ter wild-type (WT) mice. Static exposures were conducted in 29-l gas chromatographic jars. Animals were exposed to toluene (0, 1000, 2000 or 4000 ppm; n=8-12, each) for 30 min, and immediately after, tested in one of the anxiety paradigms. Motor coordination was evaluated in the rota-rod test in independent groups of mice. Toluene produced a dose-dependent decrease in anxiety-like levels in both anxiety paradigms and in both the strains. However, toluene exerted its effects at lower concentrations in KO mice than in the WT strain. These results cannot be attributed to a decrease in motor coordination since all the animals behaved similarly in the rota-rod test, regardless of the treatment. To discard any inherent difference in the nociception threshold between strains, mice were tested in the hot plate immediately after being exposed to either air or toluene. Toluene increased nociception in a similar fashion in both the strains. Our results suggest that 5-HT(1B) KO mice are more sensitive to those of toluene's actions related to anxiety, but not to those related with motor coordination or nociception. Data are discussed in terms of toluene's mechanisms of action and on differences between WT and KO animals.

Gender differences in the cardiovascular responses to morphine and naloxone in spinal rats.

Putative gender differences in opiate cardiovascular effects were evaluated in spinal rats. After a 4-h exposure to a single dose of morphine (30 mg/kg, i.v.), abstinence was precipitated by naloxone (0.03-3 mg/kg, i.v.). Morphine produced a long-lasting bradycardia and a transient increase in arterial pressure that was similar in both genders. Thereafter, blood pressure decreased both in males and females. Naloxone precipitated a similar dose-dependent heart rate increase in both sexes and a gender-dependent increase in blood pressure. This sex difference appeared in the shape of the response. Prazosin (0.2 mg/kg), prior to naloxone, reduced the pressor response in all animals, suggesting a similar participation of the noradrenergic system in both genders. The present results extend to acute dependence the notion of a sex-dependent differential effect of morphine. The need to consider gender as a factor when studying the effects of opioids is highlighted.

Blockade of the anxiolytic-like action of ipsapirone and buspirone, but not that of 8-OH-DPAT, by adrenalectomy in male rats.

The effect of the 5-HT1A agonists ipsapirone (5 mg/kg), buspirone (5 mg/kg) and 8-OH-DPAT (0.5 mg/kg) on experimental anxiety was examined in sham-operated, adrenalectomized and adrenally demedullated male rats. The animal model of anxiety used was the defensive burying test. At the doses selected, all 5-HT1A compounds produced an anxiolytic-like action by reducing the burying behavior in both sham-operated and demedullated rats. However, in adrenalectomized subjects, while 8-OH-DPAT still reduced burying behavior, ipsapirone and buspirone lost their action. Data suggest that adrenocortical secretions play a role in the anxiolytic-like actions of buspirone and ipsapirone, but not in those of 8-OH-DPAT. Buspirone and ipsapirone also produced a reduction in burying behavior latency in sham-operated animals that was not observed in adrenalectomized or adrenally demedullated rats. These data suggest that adrenaline may be participating in the action of these compounds on the burying behavior latency. Present findings support possible direct relationships between the stimulation of 5-HT1A receptors and adrenal secretions.

Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

Previous studies have shown that toluene, which is commonly abused, depresses neuronal activity and causes behavioral effects in both animals and man similar to those observed for ethanol. In this study, the oocyte expression system was used to test the hypothesis that toluene, like ethanol, inhibits the function of ionotropic glutamate receptors. Oocytes were injected with mRNA for specific N-methyl-D-aspartate (NMDA) or non-NMDA subunits and currents were recorded using conventional two-electrode voltage clamp. To enhance the low water solubility of toluene, drug solutions were prepared by mixing toluene with alkamuls (ethoxylated castor oil) at a 1:1 ratio (v:v) and diluting this mixture to the appropriate concentration with barium-containing normal frog Ringer solution. Alkamuls, up to 0.1%, had no significant effects on membrane leak currents or on NMDA-induced currents. Toluene, up to approximately 9 mM, had only minor effects on membrane leak currents but dose-dependently inhibited NMDA-mediated currents in oocytes. The inhibition of NMDA receptor currents by toluene was rapid, reversible and the potency for toluene's effects was subunit dependent. The NR1/2B subunit combination was the most sensitive with an IC50 value for toluene-induced inhibition of 0.17 mM. The NR1/2A and NR1/2C receptors were 6- and 12-fold less sensitive with IC50 values of 1.4 and 2.1 mM, respectively. In contrast, toluene up to approximately 9 mM did not inhibit kainate-induced currents in oocytes expressing GluR1, GluR1(+)R2 or GluR6 subunits. These results suggest that some of the effects of toluene on neuronal activity and behavior may be mediated by inhibition of NMDA receptors.

Tachyphylaxis and sensitization to nicotine-induced tachycardiac and pressor effects after nicotine infusions.

This work examined the effects of nicotine on mean arterial pressure and heart rate in non-anesthetized spinal rats. Nicotine (200 mg/kg) was administered as a single bolus, as infusions lasting 7.5, 15 or 30 min, and as a post-infusion bolus. A nicotine bolus increased pressure and rate. These effects were less marked as the rate of infusion decreased. The infusions affected differentially the effects of a subsequent bolus. Thus, while tachycardia was decreased, the blood pressure rise was increased. An initial transient bradycardia was observed after bolus administration, but not during infusions; this effect was unchanged after post-infusion boluses. Pharmacological analysis indicated that tachycardia and bradycardia were predominantly due to ganglionic stimulation, while adrenal and sympathetic nerve catecholamine release played a major role in the pressor response. These results indicate that slow nicotine infusions do not induce tachyphylaxis for all of the cardiovascular effects of a subsequent bolus, and that development of acute tolerance appears to depend on the mechanism of action of the response.

Further evidence that naloxone acts as an inverse opiate agonist: implications for drug dependence and withdrawal.

To test if naloxone behaved as an inverse agonist rather than as an antagonist we evaluated its responses in guinea-pig ilea with and without morphine (480 nM, 24 h). In control ilea, naloxone (100 nM) had no effect. In morphine-treated ilea, naloxone as a bolus, but not as an infusion, elicited an abstinence response. Preadministration of naloxone blocked the response to subsequent administrations. Similarly, naloxone failed to produce an abstinence response in ilea pretreated with kappa compounds (bremazocine, U50488 or xorphanol 100 nM) or with kinase inhibitors (H7 or H8 30 microM). These findings can be interpreted in the light of the two-state receptor model if naloxone behaves as an inverse agonist: Incubation with morphine increased the active state of receptors making them susceptible to the inverse agonist (naloxone); exposure to naloxone favored the inactive conformation making them insensitive to further administration of naloxone; kappa compounds behaved as antagonists preventing the response to naloxone; and kinase inhibitors interfered with the active conformation making the system insensitive to naloxone. According to this model, dependence can be viewed as an overexpression of the active receptors and withdrawal as an abrupt change from the active to the inactive state.

Effect of temperature on opioid dependence and on the abstinence response in the isolated guinea pig ileum.

This paper explores, in the isolated guinea-pig ileum, the effects of temperature on the acute development of opioid dependence and on the precipitation of the abstinence response, using as reference the effect of temperature on the response to a standard nicotine concentration. Additionally, the influence of temperature on acute morphine neurodepression was examined. Three experimental groups were included. In the first, the bath temperature was adjusted and maintained along the experimental session (2.5 h) at one of the following values: 28, 32, 36 or 40 degrees C. In the second, the different values of bath temperature were applied only during the period of morphine exposure before testing the abstinence response at 36 degrees C. In the third, all segments were initially incubated at 36 degrees C for 1 h, and afterwards, abstinence and the nicotine response were elicited at the different temperatures mentioned. In all the series, a single challenge naloxone dose (3.1, 10, 31, 100, 316, 1000 or 3160 nM) was administered after 1h of morphine and complete naloxone concentration-response curves were obtained. The abstinence response was expressed as a percentage of the nicotine reference response. All segments showed robust nicotine responses at all the experimental protocols tested indicating that, at the temperature range studied, the contractile mechanisms were impaired. This study showed that changes in bath temperature modify the magnitude of acute morphine neurodepression, and of the abstinence response but did no affect the development of acute opioid dependence. These data, along with several lines of evidence, strongly suggest that acute neurodepression, the development of opiate dependence and antagonist-precipitated abstinence are separable. Results are discussed on the basis of drug-receptor interactions.

Cardiovascular effects of different schedules of nicotine administration on spinal rats: influence of pentobarbital.

This work examined the cardiovascular effects of different schedules of i.v. nicotine administration with respect to doses and timing in non-anesthetized and pentobarbital-treated spinal rats. For this purpose, complete nicotine dose-response curves were made for mean arterial pressure and heart rate. In non-anesthetized spinal rats, tachyphylaxis was not found for the pressor effects; moreover, consecutive doses, given at short intervals, produced additive actions. Furthermore, nicotine produced a biphasic heart rate response: an initial and brief bradycardia followed by a longer lasting tachycardia. In pentobarbital-treated rats, the sensitivity of the cardiovascular system to nicotine was decreased; in these rats, consecutive nicotine doses did show tachyphylaxis for the pressor and tachycardiac responses. The present series of experiments, using different schedules of administration for single and consecutive nicotine doses, demonstrated opposite tachyphylactic effects in non-anesthetized and in pentobarbital-treated spinal rats.

Quantitative analysis of concentration-response curves for precipitated abstinence responses in the opiate-dependent guinea pig ileum.

Our group has introduced a theoretical model of drug-receptor-effector interactions that can account for most of the kinetic aspects of opiate dependence and abstinence. The present study analyzes the experimental concentration-response curves for precipitated abstinence responses in the isolated guinea pig ileum and compares them with the curves generated by the mathematical model. Two experimental series were included: in the first, the effects of morphine exposure time on dependence development were analyzed; in the second, the effects of morphine concentration were studied. The results show that both the exposure time and morphine concentration determine the magnitude of precipitated abstinence responses. Dependence can be detected after exposure times to opiates as short as 7.5 min. The concentration-response curves for abstinence responses precipitated by naloxone in ilea exposed to morphine for different periods showed: 1) an initial increase in Emax; 2) a parallel shift to the left in the position of the abstinence curves as dependence develops; and 3) an asymptotic limit to the displacement on the left. The results presented here fit well with theoretical predictions. Practical and theoretical implications are discussed.

Acute opioid dependence in the cardiovascular system of the spinal rat.

This article analyzes and characterizes the early development of opioid dependence in the cardiovascular system of the spinal rat. The main part of the study deals with changes in mean arterial pressure and heart rate induced by naloxone (NLX) in spinal rats pretreated with single doses of morphine. The results show that the cardiovascular system of the spinal rat is highly sensitive to the abstinence-precipitating actions of NLX after a single dose of morphine. Dose-response curves for precipitated abstinence evaluated as changes in mean arterial pressure and heart rate show a gradual increase in maximum followed by a progressive shift to the left as dependence progresses. Cardiovascular abstinence is mostly mediated by catecholaminergic systems. NLX also precipitates noncardiovascular signs of abstinence. Interestingly, morphine-free spinal rats gave some abstinence-like responses to NLX, probably because of endogenous release of opioids. The spinal rat seems to be a valuable system for a rapid quantitative pharmacological characterization of the abstinence-precipitating actions of opioid antagonists and for the study of the changes that occur during the acute development of opioid dependence.

Gastrointestinal effects of 5-hydroxytryptamine and related drugs.

This paper deals with the effects of 5-hydroxytryptamine (5-HT; serotonin) and related drugs on the gastrointestinal tract (GIT). The nomenclature and classification of 5-HT receptors, as well as their putative role in the GIT are updated in this review. Besides its effects on the cardiovascular system, which have been extensively described, several lines of evidence suggest a role for 5-HT in regulating gastrointestinal functions. 5-HT is present in the gastrointestinal tissues, and can elicit contraction or relaxation by activation of a wide variety of mechanisms and receptors. At least four main types of receptors (5-HT1, 5-HT2, 5-HT3 and 5-HT4) have been described and all the four types seem to influence the GIT. In this respect, the 5-HT2, and in some cases the 5-HT1 receptors, appear to be present on the gastrointestinal smooth muscle, while 5-HT3 and 5-HT4 are mainly neuronal.

Human aldrin poisoning

Sixteen patients acutely poisoned with aldrin were examined to evaluate a possible correlation between serum aldrin and diedrin levels and clinical complaints. The patients were classified as having mild (N = 8), moderate (N = 5) or severe (N = 3) poisoning according to clinical symptoms. Concentrations of less than 20 µg/l were usually associated with mild poisoning, which involved complaints such as nausea, vomiting and epigastric pain, whereas concentrations of 100 to 200 µg/l were considered to represent moderate intoxication and were associated with nausea, vomiting, epigastric pain, headache, dizziness, and convulsions. Sever or fatal cases were associated with levels above 700 µg/l. Taken together,these results suggest that serum aldrin diedrin levels can be used as indicators of clinical prognosis after acute poisoning with these insecticides and that convulsions could suddenly occur even in the absence of prodronal signs or symptoms

A methodological basis for improving the reliability of measurements of opiate abstinence responses in the guinea-pig ileum made dependent in vitro.

The purpose of this study was to find a neurogenic response of the isolated guinea-pig ileum that could serve as an internal standard to normalize abstinence responses, which are also neurogenic, during opiate dependence produced in vitro. The internal standard is required because of baseline variability in these responses of the ileum and a time-dependent decay in neuroeffector responsiveness with prolonged incubation. Systematic studies were made of the variability in the responses to neurogenic stimulation by 1) electrical field stimulation, 2) nicotinic stimulation, and 3) precipitation of opiate abstinence with opiate antagonists as well as studies of the time-dependent decay in responsiveness with prolonged incubation. The three neurogenic responses show covariation but the best correlation is found between the nicotinic and the abstinence responses. The nicotinic system presents a pharmacological sensitivity to specific acute opiate action and also shows an improving correlation with abstinence which develops with the progression of dependence. This correlation tends to a direct linear relation with a slope approaching 1.0. The nicotinic response of the ileum seems to be a valid internal control to normalize its abstinence responses after incubation with opiates for different intervals of time.

Human aldrin poisoning.

Sixteen patients acutely poisoned with aldrin were examined to evaluate a possible correlation between serum aldrin and dieldrin levels and clinical complaints. The patients were classified as having mild (N = 8), moderate (N = 5) or severe (N = 3) poisoning according to clinical symptoms. Concentrations of less than 20 micrograms/l were usually associated with mild poisoning, which involved complaints such as nausea, vomiting and epigastric pain, whereas concentrations of 100 to 200 micrograms/l were considered to represent moderate intoxication and were associated with nausea, vomiting, epigastric pain, headache, dizziness, and convulsions. Severe or fatal cases were associated with levels above 700 micrograms/l. Taken together, these results suggest that serum aldrin and dieldrin levels can be used as indicators of clinical prognosis after acute poisoning with these insecticides and that convulsions could suddenly occur even in the absence of prodromal signs or symptoms.