No increase in carcinogen-DNA adducts in the lungs of monkeys exposed chronically to marijuana smoke.

Rhesus monkeys exposed to marijuana smoke either 7 or 2 days/weeks (HI and LO groups, respectively), or ethanol-extracted marijuana smoke for 7 days/week (EM) or sham treatment (SH) for 1 year were sacrificed 7 months following the last exposure. Pulmonary levels of carcinogen-DNA adducts were determined. Although mean or median adduct levels were not statistically different, 15 of 22 adduct measures were highest in the EM group and lowest 12 of 22 times in the SH group. The levels of aromatic carcinogen-DNA adducts seem no higher in the lungs of animals exposed to marijuana smoke than in untreated animals. Ethanol-extracted marijuana may have effects greater than marijuana itself.     

Hematological changes in rats chronically exposed to oral aluminum

This study was aimed to investigate the effects of the long-term oral exposure to aluminum sulfate on hematological parameters in rats. For this purpose, 24 adult female Wistar rats were divided in three groups with 8 animals each (control, citrate, and citrate plus aluminum groups). Rats from control and citrate groups had free access to tap water and to a sodium citrate solution (35 mM), respectively. Rats from citrate plus aluminum group received, as unique source of liquid, an aluminum sulfate solution (30 mM) diluted in the above-mentioned sodium citrate solution, ad libitum. After the treatment period (18 months), aluminum-exposed rats showed a significant decrease in the number of red blood cells, blood hemoglobin concentration and hematocrit when compared to rats from the control group. Serum iron levels were also significantly lower in citrate plus aluminum group, whereas total iron binding capacity did not change after citrate plus aluminum exposure. Erythrocyte thiobarbituric acid-reactive substances (TBARS) and nonprotein thiols (NPSH) levels, erythrocyte osmotic fragility and hepatic delta-aminolevulinic acid dehydratase (delta-ALA-D) activity did not change after treatment with citrate plus aluminum. Conversely, aluminum exposure increased delta-ALA-D activity in bone marrow. The present results indicate that long-term oral exposure to low doses of aluminum sulfate promotes alterations on erythrocyte parameters in rats, probably as a consequence of alterations in the iron status. In addition, although the details of the underlying mechanism remain unclear, our study reports, for the first time, a stimulatory effect of chronic aluminum exposure on bone marrow delta-ALA-D activity.     

Vulnerability of gastric mucosa to prednisolone in rats chronically exposed to cigarette smoke

We examined gastric mucosal vulnerability in a rat model of chronic obstructive pulmonary disease (COPD). Male Wistar rats were exposed to cigarette smoke for 12 weeks (CSE rats), and on the last 4 days of exposure, prednisolone was given to induce gastric mucosal injury. Histopathology, pulmonary function, arterial blood gases, and levels of lipid peroxides (LPO), prostaglandin E(2) (PGE(2)), hypoxia-inducible factor 1 alpha subunit (HIF-1alpha), and vascular endothelial growth factor (VEGF) in gastric mucosa were examined. We also tested the effect of rebamipide on prednisolone-induced gastric lesions. In CSE rats, although no gastric lesions were detected, LPO, PGE(2), HIF-1alpha, and VEGF levels were higher than in control rats. Prednisolone induced gastric hemorrhagic lesions more readily in CSE rats than controls, with concomitant decrease in PaO(2) and increased levels of LPO, HIF-1alpha, and VEGF. Rebamipide reversed gastric lesions without affecting any parameters examined. CSE rats were found to be a useful animal model of COPD, and COPD appeared to render the gastric mucosa vulnerable to prednisolone.     

Behavioral and neurochemical changes in rats simultaneously exposed to manganese and lead

Groups of rats were exposed simultaneously to manganese chloride (3 mg Mn²⁺/ml water) through drinking water and lead acetate intraperitoneally at dosages of 5.0, 8.0 and 12.0 mg Pb²⁺/kg daily for a period of 14 days. The magnitude of changes in the behavioral pattern, contents of biogenic amines and accumulation of lead in the brain of rats simultaneously exposed to the two metals was significantly greater than observed in rats after exposure to either of the metals alone. A definite dose-response relationship was, however, noticed only with the changes in the motoractivity, norepinephrine, 5-hydroxytryptamine levels and in the accumulation of lead in rats simultaneously exposed to manganese and lead. The lowering in the contents of norepinephrine after combined treatment was found to be related with the decrease in the motoractivity in the rats. The exact role of depression in the levels of dopamine and 5-hydroxytryptamine in inducing marked impairment in learning ability and increased aggressive behavior in rats after the combined exposure to manganese and lead could not be ascertained. The overall analysis of the data indicated that the simultaneous exposure to manganese and lead, particularly with highest dose of the latter, may produce serious derangements in the behavioral pattern and levels of biogenic amines in the brain of rats.     

Pulmonary pathologic changes in rats exposed to marihuana smoke for 1 year.

Exposure of rats to marihuana or placebo smoke for periods up to 365 days was performed with an automatic inhalator. Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) concentrations in the marihuana smoke were similar to those inhaled by man and were presented to the rats in a 50-ml puff volume of 2-sec duration and a 30-sec exposure interval followed by a 30-sec period of fresh air each minute (1 puff/min). By varying the number of puffs from three simultaneously smoked marihuana cigarettes (0.9 to 1.2% Δ⁹-THC), 8 to 10 Fischer rats simultaneously received a single daily Δ⁹-THC dose of 0.4, 0.8 or 1.5 mg/kg, 6–7 days per week for 365 days. All treatment groups contained 30 males and 30 females except for the high-dose group which had 50 males and 50 females. At each of the lower doses $\text{2}\text{60}$ (3%) of the animals died while at the higher dose $\text{18}\text{108}$ (17%) died. Twenty-five percent ($\text{15}\text{60}$) of the placebo-smoked rats died primarily from carbon monoxide poisoning. No sham-smoked rats died. In deceased marihuana-smoked rats, organ congestion and focal petechial hemorrhages in the brain suggested circulatory failure. In contrast with an earlier 87-day inhalation study, pulmonary irritation progressed beyond a dose-related focal alveolitis or pneumonitis with the accumulation of yellow-brown alveolar macrophages admixed with a few neutrophils and mononuclear cells, to a spectrum of more pronounced inflammatory and focal proliferative changes. The development of focal granulomatous inflammation in the lung with giant cell forms of macrophages and cholesterol-like clefts were striking new developments in the marihuana-smoked rats, especially since these effects were dose related and nonreversible after a 30-day recovery period.     

Cadmium accumulation in the lung, liver and kidney of mice and rats chronically exposed to cigarette smoke.

Cigarette smoking constitutes a major source of cadmium exposure via inhalation in man. To determine how smoke exposure affects the organ distribution and accumulation of cadmium, male C57B1 mice and Sprague-Dawley rats were exposed daily for 52-60 consecutive weeks to mainstream smoke from the University of Kentucky reference cigarettes (2R1) in a nose-only exposure system. Exposed mice and rats averaged blood carboxyhemoglobin values of 17.7 and 7.2%, and a daily total particulate matter (TPM) dose of 7.2 and 3.2 mg kg-1 body wt. per exposure, respectively. These results suggested effective inhalation of smoke by the animals. The tissues were acid digested and analyzed for cadmium by atomic absorption spectrometry. Cadmium levels 5-6- and 2-3-fold greater than control levels were detected in the lungs and kidneys, respectively, of exposed animals of both species. In contrast, the liver did not show increased cadmium levels in exposed mice or rats. The data suggest that low-dose chronic inhalation exposure to cigarette smoke leads to highest cadmium accumulation in the lung, followed by the kidney, with minimal effects on cadmium levels in the liver.     

Tolerance to the behavioral and neurochemical effects of haloperidol and morphine in rats chronically treated with morphine or haloperidol

Summary The acute administration of morphine sulfate (79 moles/kg) or haloperidol (6.65 moles/kg) produced catalepsy and concomitant increase in striatal dopamine turnover in rats. The animals made dependent on morphine by 52 morphine injections (maintenance dose of 1056 moles/kg/day, given in four daily doses) and then tested during 3 days of withdrawal from morphine, showed tolerance to the cataleptic and the neurochemical effects of morphine as well as those of haloperidol. That tolerance was not seen after 14 days of withdrawal from morphine. The animals chronically treated with haloperidol for 12 days (maintenance dose of 53.2 moles/kg/day, given in two daily doses) and then tested 72 h after last haloperidol injection, did not show tolerance to the cataleptic or the neurochemical effect of haloperidol or morphine. These results suggest that dopaminergic systems underlying motor coordination and regulation of the neurotransmitter synthesis are among those susceptible to narcotic action and to the process of tolerance development during aarcotic dependence.     

Characterization of biochemical,functional and structural changes in mice respiratory organs chronically exposed to cigarette smoke

Abstract

Female C57BL/6 mice were exposed to mainstream cigarette smoke at 600?μg WTPM/L, 4?h/day and 5 days/week for up to 52 weeks. At 26, 52 and 65 weeks (52 weeks of exposure plus 13 weeks of no exposure), lungs were assessed for inflammation, function, histopathology and morphometry. Structural changes were observed and accompanied by altered lung function at 26 and 52 weeks (e.g. increase of static compliance and hysteresis, and decrease of elastance). Lung morphometry quantified significant increase in airspace enlargement at 52 weeks. Chronic smoke exposure induced inflammation in respiratory organs, e.g. mixed inflammatory cell infiltrates, perivascular lymphocyte infiltrates and pigmented alveolar macrophages in the lungs. Minimal or mild alveolar emphysema was diagnosed in 70% by 26 weeks or 80% by 52 weeks. After 13 weeks of recovery, most biochemical, histopathological and morphometrical alterations were restored, while emphysema was observed to persist at 18% incidence by 65 weeks. In conclusion, the employed exposure conditions induced emphysematous changes in the lungs, accompanied by altered lung function and morphological/histopathological changes. Following the 13 weeks of no exposure, morphological changes persisted, although some functional/biochemical alterations regressed.     

Repeated measures designs in behavioral toxicology: application to chronic marijuana smoke exposure

This paper discusses the application of repeated measures methods in the statistical analysis of an experiment in behavioral toxicology. The chronic marijuana smoke exposure study conducted at the National Center for Toxicological Research is used for an example of the types of problems that one encounters in analyzing these types of studies. In particular, the standard univariate analysis most frequently used for repeated measures analyses has some very restrictive assumptions on the form of the covariance matrices. These assumptions are not met in the example discussed and are rarely met in many other problems. Other possible models for analyzing repeated measures when these assumptions are not met are presented and discussed. Other problems specific to the chronic marijuana smoke exposure study that may occur in similar type studies are presented. These include pooling the experimental units into groups with comparable baselines, choosing a function of the measures to be analyzed, dealing with a large data set with many observation times and missing data, unequal group sizes and different designs for different subsets of the experimental animals. The standard univariate repeated measures analysis was chosen to analyze the data even though the violations of the covariance assumptions may lead to finding differences that do not exist (Type I or false-positive errors), since the other methods presented also had covariance assumptions that were not met or had low power. Use of Bonferroni-type multiple comparisons on the single degree of freedom contrasts of interest hopefully reduced the chances of these false-positive results.     

Evidence that exposure to methyl mercury during gestation induces behavioral and neurochemical changes in offspring of rats

On day 15 of gestation, pregnant Sprague-Dawley rats were orally treated by gavage with 8 mg/kg of methyl mercury (MMC). At day 1 of postnatal life the levels of MMC in whole brain of exposed pups were found to be about 100 times higher than those of saline-exposed rats, while they were near to the control values at 21 days and practically normal at 60 days of age. Behavioral experiments showed that exposure to MMC in late gestation did not affect at any tested time (14, 21 and 60 days) locomotor activity or development of ultrasonic vocalization. An increased response to a challenge dose of amphetamine was, however, detected in MMC-exposed pups at day 14. This phenomenon was no longer evident at day 21 and 60 of age. In parallel, an increased density of dopamine receptors was found in the striatum at 14, but not at 21 and 60, days of age. From these data, we tentatively suggest that a high level of MMC induces a transient phenomenon of disuse-supersensitivity of the dopaminergic system. Moreover, further evidence that acute MMC exposure during prenatal life might induce permanent disturbances in learning and memory which could be partially related to a reduced functional activity of the glutamatergic system is provided.     

Gender impacts behavioral and neurochemical adaptations in ethanol-dependent rats

Previous investigations have found gender differences in the effects of chronic ethanol exposure on ethanol withdrawal behaviors as well as GABA(A) receptor gene expression. The present investigation extended these studies with additional behavioral and neurochemical measures of ethanol dependence and withdrawal. No significant gender differences in the elevated plus-maze assessment of ethanol withdrawal anxiety behaviors were found. However, the neuroactive steroid, 3alpha,5alpha-THP, increased exploratory behavior in ethanol withdrawn female, but not male, rats. GABA(A) receptor binding assays showed potent competition of [35S]TBPS binding by 3alpha,5alpha-THP. Control females displayed a decreased affinity for 3alpha,5alpha-THP compared to control males, as evidenced by a nearly 30% increase in the IC50 value. There was no significant effect of ethanol withdrawal on 3alpha,5alpha-THP modulation of [35S]TBPS binding. However, gender differences were observed in the effects of chronic ethanol exposure on GABA(A) receptor subunit peptide levels in the hypothalamus. Female rats had a significant increase in peptide levels for the alpha2 and alpha3 but not alpha4 subunit, whereas male rats displayed a significant increase in alpha4 and alpha3 but not alpha2 subunits compared to pair-fed control levels. Chronic ethanol-induced alterations in gene expression in the hypothalamus did not coincide with previous findings in the cerebral cortex. In particular, male rats showed an increase in alpha1 subunit peptide levels in the hypothalamus, whereas significant decreases in this subunit have been observed in the cerebral cortex. Both female and male rats showed significant increases in the alpha3 subunit in the hypothalamus but not the cerebral cortex. Taken together, these studies provide additional support for gender-selective effects of chronic ethanol-elicited adaptations at the molecular level.     

Reactivity to amphetamine in perinatally undernourished rats: behavioral and neurochemical correlates

Adult rats deprived at perinatal age and then rehabilitated on balanced chow were treated with a multiple amphetamine (AMPH) schedule (2 mg/kg/48 hr) and submitted, on days of injections, to an open-field test. Throughout 11 sessions, deprived rats showed a progressive increase of locomotor activity as compared with controls. Stereotyped activity evaluated during the AMPH treatment did not differ between control and deprived animals. No differences were detected in basal values of the dopaminergic function measured in naive control and deprived animals. By the end of the multiple AMPH treatment, a reduction of striatal DA and DOPAC levels together with a lower apparent DA turnover rate was detected in deprived animals. Besides, DA receptor binding was significantly increased in striatum from deprived rats as compared with controls. These results demonstrate that a repeated AMPH treatment, that was unable to alter the normal behavior of control rats, produced in early undernourished animals a progressive sensitization to AMPH effects, in addition to significant changes in the striatal dopaminergic function.     

Ultrastructural changes of tracheal epithelium and alveolar macrophages of rats exposed to mosquito coil smoke

The volatile organic compounds of mosquito coil smoke were analysed by a combination of gas chromatography and mass spectrometry. 67 volatile organic compounds were detected and 46, including allethrin, phenol, benzene, toluene, xylene, etc. could be identified. Allethrin, a common name for (+/-)-3-allyl-2-methyl-4-oxocyclopent-2-enyl (+/-)-cis,trans-chrysanthemate, is a synthetic pyrethroid, which is used as an insecticide or repellent in subtropical countries. The vapors also contain other aromatic and aliphatic hydrocarbons which are combustion products of other mosquito coil constituents, including wood dust, coconut flour and other fillers and dyes. An exposure to the mosquito coil smoke for 60 days (8 h per day, 6 days per week) resulted in a focal deciliation of the tracheal epithelium, metaplasia of epithelial cells, and morphological alterations of the alveolar macrophages of exposed rats.     

Bone metabolism of male rats chronically exposed to cadmium

Recently, based on a female rat model of human exposure, we have reported that low-level chronic exposure to cadmium (Cd) has an injurious effect on the skeleton. The purpose of the current study was to investigate whether the exposure may also affect bone metabolism in a male rat model and to estimate the gender-related differences in the bone effect of Cd. Young male Wistar rats received drinking water containing 0, 1, 5, or 50 mg Cd/l for 12 months. The bone effect of Cd was evaluated using bone densitometry and biochemical markers of bone turnover. Renal handling of calcium (Ca) and phosphate, and serum concentrations of vitamin D metabolites, calcitonin, and parathormone were estimated as well. At treatment with 1 mg Cd/l, corresponding to the low environmental exposure in non-Cd-polluted areas, the bone mineral content (BMC) and density (BMD) at the femur and lumbar spine (L1-L5) and the total skeleton BMD did not differ compared to control. However, from the 6th month of the exposure, the Z score BMD indicated osteopenia in some animals and after 12 months the bone resorption very clearly tended to an increase. The rats' exposure corresponding to human moderate (5 mg Cd/l) and especially relatively high (50 mg Cd/l) exposure dose- and duration-dependently disturbed the processes of bone turnover and bone mass accumulation leading to formation of less dense than normal bone tissue. The effects were accompanied by changes in the serum concentration of calciotropic hormones and disorders in Ca and phosphate metabolism. It can be concluded that low environmental exposure to Cd may be only a subtle risk factor for skeletal demineralization in men. The results together with our previous findings based on an analogous model using female rats give clear evidence that males are less vulnerable to the bone effects of Cd compared to females.     

Clinical and biochemical changes in chronically exposed organophosphate workers

Health effects of occupational organophosphate exposure were investigated by subjecting 22 workers chronically exposed to an organophosphate pesticide, fenthion (O,O-dimethyl-O-(4-methylmercapto-3-methylphenyl)-phosphorothioate) to clinical evaluation, estimation of serum cholinesterase, serum alkaline phosphatase (SAP), serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT). The mean age of the workers was 31 years and the mean duration of fenthion exposure 8.2 years. Headache (59%), giddiness (50%), ocular symptoms (27%) and paresthesia (18%) were the commonest symptoms. Serum acetylcholinesterase and butyrylcholinesterase levels were significantly lower than in controls. After withdrawing the workers from organophosphate exposure for 3 weeks, the follow-up study revealed absence of transient symptoms. There was no change in their neurological status, and serum acetylcholinesterase and butyrylcholinesterase levels were raised significantly, whereas the other serum enzymes showed no significant change. For monitoring of occupational organophosphate exposure, the importance of both clinical and biochemical parameters is emphasised.     

Carbamazepine normalizes the altered behavioral and neurochemical response to stress in benzodiazepine-withdrawn rats

Rats chronically treated with diazepam (2 mg/kg per day, i.p.) for 21 days were tested 96 h after the last injection in both the forced swim test (inescapable stress) and in an active avoidance test (escapable stress). The influence of carbamazepine (7.5 mg/kg, i.p.) administered 25 min prior to each behavioral task was investigated. Withdrawn animals showed a reduced time spent in immobility in the forced swim test and an enhanced latency to escape in the active avoidance test. Both behavioral effects were normalized by a single carbamazepine administration. An additional experiment was performed to investigate the effect of a forced swim experience on cortical chloride uptake following GABA (γ-aminobutyric acid) stimulation 96 h after diazepam withdrawal, and the influence of a single administration of carbamazepine on these effects. An increased chloride uptake was observed in vehicle-treated rats but not in diazepam-withdrawn animals following the swimming experience. Carbamazepine pretreatment enhanced chloride uptake after diazepam withdrawal but did not modify chloride flux in stressed or unstressed vehicle-treated rats. These results support the hypothesis that diazepam withdrawal affects the ability to develop adaptive responses to stress and that carbamazepine can normalize such an alteration.     

Breathhold duration and response to marijuana smoke

Marijuana smokers are frequently observed to hold the smoke in their lungs for prolonged periods (10-15 sec) apparently in the belief that prolonged breathholding intensifies the effects of the drug. The actual influence of breathhold duration on response to marijuana smoke has not been studied. The present study examined the effects of systematic manipulation of breathhold duration on the physiological, cognitive and subjective response to marijuana smoke in a group of eight regular marijuana smokers. Subjects were exposed to each of three breathhold duration conditions (0, 10 and 20 sec) on three occasions, scheduled according to a randomized block design. A controlled smoking procedure was used in which the number of puffs, puff volume and postpuff inhalation volume were held constant. Expired air carbon monoxide levels were measured before and after smoking to monitor smoke intake. Typical marijuana effects (increased heart rate, increased ratings of "high" and impaired memory performance) were observed under each of the breathhold conditions, but there was little evidence that response to marijuana was a function of breathhold duration.     

Prenatal cocaine exposure alters behavioral and neurochemical sensitization to amphetamine in adult rats

This study examined the neurochemical correlates of amphetamine (AMPH)-induced behavioral effects in prenatally saline (PSAL)-exposed or cocaine (PCOC)-exposed male rats. Pregnant Long-Evans rats received saline or saline containing cocaine hydrochloride (20 mg/kg s.c., b.i.d.) from gestational days 15-21. Animals were left with their biological mothers. Adult offspring were exposed to daily saline or AMPH (0.5, 1.5, or 5 mg/kg, i.p.) injections for 7 days. Behaviors were recorded in an open field during the first hour post-injection. PCOC rats did not exhibit behavioral anomalies during habituation to injection-stress or placement in the open field. PCOC rats displayed significant alterations in stereotyped responses to acute or intermittent exposure to various doses of AMPH. Within 48 h of the final testing day, striatal tissue was obtained from these animals and electrically-evoked [3H]acetylcholine (ACh) release was measured from striatal slices. Superfusion of tissue slices with various concentrations of AMPH (1-1000 nM) produced dose-dependent inhibition of ACh release in both PSAL and PCOC rats repeatedly injected with saline as adults. However, AMPH-induced inhibition of ACh release was decreased in PCOC rats repeatedly injected with AMPH as adults. At 5 mg/kg AMPH, PCOC rats exhibited increased mortality compared to PSAL rats. These data suggest that PCOC exposure produces long-lasting alterations in nigrostriatal transmission and behaviors mediated by this system.     

In vitro neuronal and vascular responses to 5-HT in rats chronically exposed to MDMA.

1. This study examined the effects of chronic exposure of rats to 3,4-methylenedioxymethamphetamine (MDMA) on [(3)H]5-hydroxytryptamine ([(3)H]5-HT) re-uptake into purified rat brain synaptosomes, 5-HT-induced isometric contraction of aortic rings and [(3)H]5-HT re-uptake into rat aorta. 2. Rats were administered MDMA (20 mg kg(-1) i.p.) twice daily over 4 days. One, 7, 14 or 21 days post treatment, whole brain synaptosomes and descending thoracic aortic rings were prepared for investigation. 3. Chronic MDMA treatment significantly reduced the maximum rate (V(max)) of specific high-affinity [(3)H]5-HT re-uptake 1 day after treatment and for up to 21 days post-final administration of MDMA. Direct application of MDMA (100 microM) abolished synaptosomal re-uptake of [(3)H]5-HT in vitro. 4. Chronic MDMA administration significantly reduced the maximum contraction (E(max)) to 5-HT at 1 and 7 days after treatment, but not at 14 or 21 days. 5. Chronic MDMA administration had no effect on sodium-dependent [(3)H]5-HT re-uptake into aorta 1 day after treatment, nor did 100 microM MDMA have any direct effect on [(3)H]5-HT uptake into aortic rings in vitro. 6. These results show, for the first time, an altered responsiveness of vascular tissue to MDMA after chronic administration. In addition, they demonstrate a difference in the sensitivity of central and peripheral 5-HT uptake systems to chronic MDMA exposure, and suggest that the action of MDMA in the cardiovascular system does not arise from a direct effect of MDMA on peripheral 5-HT transport.