Inhalation carcinogenicity of 1,1,1-trichloroethane in rats and mice

Abstract

Carcinogenicity of 1,1,1-trichloroethane (TCE) was examined by an inhalation exposure of F344 rats and BDF1 mice of both sexes to TCE at 0, 200, 800 or 3200?ppm for 6?h/d, 5?d/week for 104 weeks. In male rats, the incidences of bronchiolo-alveolar adenomas and peritoneal mesotheliomas were significantly increased in the 800 and 3200?ppm-exposed groups, respectively. The incidence of bronchiolo-alveolar adenomas in the 3200?ppm-exposed groups exceeded the range of historical control data in the Japan Bioassay Research Center. In female rats, the tumor incidences were not increased in any organs of the TCE-exposed groups. In male mice, a significant positive trend with dose was shown for incidences of bronchiolo-alveolar carcinomas, combined incidences of bronchiolo-alveolar adenomas/carcinomas and hepatocellular adenomas. The incidence of Harderian gland adenomas was significantly increased in the 3200?ppm-exposed group, and malignant lymphomas of spleen at this highest dose exceeded the range of historical control data. In female mice, the combined incidence of bronchiolo-alveolar adenomas/carcinomas was significantly increased in the 3200?ppm-exposed group, and the incidences of hepatocellular adenomas and combined incidences of hepatocellular adenomas/carcinomas were significantly increased in the 200, 800 and 3200?ppm-exposed groups with dose dependence except the combined incidence of hepatocellular adenomas/carcinomas in the 200?ppm-exposed group. The incidences of bronchiolo-alveolar adenomas in the 3200?ppm-exposed group and combined incidences of hepatocellular adenomas/carcinomas in the 200?ppm-exposed groups exceeded the ranges of historical control data. Thus, this study provided clear evidence of inhalation carcinogenicity for TCE in both rats and mice.     

Effects of 1,1,1-trichloroethane on the cGMP metabolism in mouse brain

Administration of 1,1,1-trichloroethane (TCE) to mice by inhalation or intraperitoneally reduced the cGMP contents of the brain stem, cerebral cortex, and vermis anterior, including the hemispheres. Following intraperitoneal administration the cGMP contents of the vermis posterior and hippocampus were also reduced. To investigate the mechanism underlying these changes, the effects of TCE on brain guanylate cyclase (GC) and phosphodiesterase (PDE) activities were examined after intraperitoneal administration in mice. The basal GC activities in the particulate and soluble fractions of the homogenates of the cerebellum, brain stem and cerebral cortex were not altered by TCE. In the cerebellum TCE treatment inhibited sodium azide-stimulated GC activity in the particulate and soluble fractions, while in the brain stem it enhanced the particulate GC activity induced by Ca2+. TCE treatment increased the rate of cGMP hydrolysis in the cerebral cortex and this was further accelerated by addition of Ca2+. Ca2+ also increased the rate of cGMP hydrolysis in the brain stem. However, in the cerebellum TCE enhanced the Ca2+-independent PDE activity as well as the enzyme activity in the presence of Ca2+ and exogenous calmodulin. These results indicate that the reduction of the cGMP content in the brain stem and cerebral cortex in vivo on exposure to TCE is due to changes in the rate of cGMP hydrolysis. In the cerebellum the TCE reduced cGMP content may be regulated by increased rate of cGMP hydrolysis as well as effects on the guanylate cyclase.     

Effects of 1,1,1-trichloroethane on synaptosomal calcium accumulation in mouse brain

Addition of 1,1,1-trichloroethane (TCE), in vitro to synaptosomes isolated from the mouse cerebellum and cerebral cortex inhibited the slow phase of K+-stimulated 45Ca2+ influx and the net 45Ca2+ influx (delta k). In the brain stem, however, TCE increased the fast and slow phases of calcium uptake under depolarizing conditions and also delta k. The non-depolarized calcium influx was not altered by TCE added in vitro. Two hours after injection of TCE (2.4 g/kg) the calcium accumulation in the presence of high K+ was lowered in the cerebellar synaptosomes, while it was increased in brain stem synaptosomes. TCE administered in vivo did not alter the calcium influx into cerebrocortical synaptosomes nor did it affect the non-depolarization-induced 45Ca2+ influx. Thus, these data indicate that TCE may influence voltage-dependent calcium channels in mouse brain synaptosomes.     

Actions of 1,1,1-trichloroethane on the cAMP metabolism in mouse brain

Inhalation or intraperitoneal administration of 1,1,1-trichloroethane (TCE) increased the cAMP content in the brain stem in a dose-related and time-dependent fashion. TCE had no effect on the cAMP level in the cerebellum and hippocampus, but slightly reduced that in the cerebral cortex. Following intraperitoneal administration of TCE the activity of norepinephrine- and F- -activated adenylate cyclase (AC) in a brain stem homogenate was enhanced, while the serotonin-stimulated AC activity was decreased. Neither the basal AC activity nor the guanine nucleotide- and forskolin-activated enzyme was affected by TCE. TCE had no effect on the soluble cAMP-dependent phosphodiesterase activity. It is suggested that the increased cAMP content in the brain stem induced by TCE may be mediated via adrenoreceptor interaction with the guanine nucleotide-binding regulatory component, resulting in activation of the catalytic unit of the adenylate cyclase and thereby an increase in the cAMP level.     

Baclofen and cerebellar cyclic GMP levels in mice.

Baclofen (3.5--10.0 mg/kg) dose dependently decreased cerebellar cyclic GMP content in mice correlated to a reduction in locomotor activity. Pretreatment with baclofen antagonized the rise in cerebellar cyclic GMP content induced by isoniazid and picrotoxin, but did not affect the increase in cyclic GMP following administration of pentetrazole. Baclofen had little effect on the arecoline-induced changes in cerebellar cyclic GMP content. Therefore, our data support the concept of gamma-aminobutyric acid agonistic properties of baclofen at presynaptic sites.     

Discriminative stimulus effects of inhaled 1,1,1-trichloroethane in mice: comparison to other hydrocarbon vapors and volatile anesthetics

Rationale  Because the toxicity of many inhalants precludes evaluation in humans, drug discrimination, an animal model of subjective effects, can be used to gain insights on their poorly understood abuse-related effects. Objectives  The purpose of the present study was to train a prototypic inhalant that has known abuse liability, 1,1,1-trichloroethane (TCE), as a discriminative stimulus in mice, and compare it to other classes of inhalants. Materials and methods  Eight B6SJLF1/J mice were trained to discriminate 10 min of exposure to 12,000 ppm inhaled TCE vapor from air and seven mice were trained to discriminate 4,000 ppm TCE from air. Tests were then conducted to characterize the discriminative stimulus of TCE and to compare it to representative aromatic and chlorinated hydrocarbon vapors, volatile halogenated anesthetics as well as an odorant compound. Results  Only the 12,000 ppm TCE versus the air discrimination group exhibited sufficient discrimination accuracy for substitution testing. TCE vapor concentration- and exposure time-dependently substituted for the 12,000 ppm TCE vapor training stimulus. Full substitution was produced by trichloroethylene, toluene, enflurane, and sevoflurane. Varying degrees of partial substitution were produced by the other volatile test compounds. The odorant, 2-butanol, did not produce any substitution for TCE. Conclusions  The discriminative stimulus effects of TCE are shared fully or partially by chlorinated and aromatic hydrocarbons as well as by halogenated volatile anesthetics. However, these compounds can be differentiated from TCE both quantitatively and qualitatively. It appears that the degree of similarity is not solely a function of chemical classification but may also be dependent upon the neurochemical effects of the individual compounds.     

Acute motor and lethal effects of inhaled toluene, 1,1,1-trichloroethane,halothane, and ethanol in mice: Effects of exposure duration

Some acute effects of inhalation exposure to toluene, 1,1,1-trichloroethane (1,1,1-TCE), halothane, and ethanol were examined in mice. Lethality and performance on an inverted screen test of motor performance were measured following 10-, 30-, and 60-min exposures. Concentration-dependent effects were obtained on both measures for all solvents except that lethal concentrations of ethanol could not be produced under these exposure conditions. Lethality increased with longer exposures for toluene, 1,1,1-TCE, and halothane. Sensitivity to the motor effects of 1,1,1-TCE, halothane, and ethanol increased when exposure duration was increased from 10 to 30 min, with no further change with 60-min exposures. In contrast, behavioral sensitivity to toluence increased over the entire range of exposure durations. The relative lipid solubilities of the solvents correlate with potency for behavioral activity but not as well with potency for lethality. The ratio of potency for motor and lethal effects depended on the chemical studied and in some cases exposure duration. These results demonstrate that both concentration and exposure duration determine the effects of inhaled compounds, but a simple linear relationship does not exist and it depends upon the effect measured.     

Tolerance and sensitization to inhaled 1,1,1-trichloroethane in mice: results from open-field behavior and a functional observational battery

Rationale 1,1,1-Trichloroethane (TCE), a representative abused solvent, has well described acute behavioral effects in animals. Much less is known about repeated high-concentration exposures as would be encountered in inhalant abusers. Tolerance has been demonstrated in some, but not all, studies with TCE while sensitization has also been seen with other abused solvents.Objective The present study was designed to further characterize changes in the effects of repeated exposure to TCE on a variety of mouse behaviors.Methods Mice were tested using locomotor activity as well as a functional observational battery (FOB) both before and after a regimen of daily exposures to various concentrations of TCE.Results The initial locomotor effects of acute 30-min exposures to TCE were biphasic with concentration-dependent increases in activity at lower concentrations and decreases observed at higher concentrations. The profile of acute effects as measured by the FOB included changes in posture, decreased arousal, disturbances in gait, delayed righting reflexes, and decreased sensorimotor reactivity. Animals were then divided into five groups and exposed 30 min/day to either air or one of four concentrations of TCE (2,000, 6,000, 10,000, or 13,300 ppm) for 15 consecutive days. The TCE concentration used primarily affected the magnitude of change, not whether tolerance or sensitization occurred. Tolerance developed on the measures of forelimb grip strength, inverted screen, and number of rears. Conversely, sensitization developed to measures of locomotor activity.Conclusion Depending on the behavioral measure, both tolerance and sensitization can occur in mice with repeated exposure to TCE. Both of these phenomena are characteristic of drugs of abuse.     

Effects of manganese on cyclic GMP levels in the rat ductus deferens

Summary Mn²⁺ and other divalent cations (Me²⁺) are capable of preventing contractile responses of smooth muscle to various stimuli. The effects of Mn²⁺, of other inhibitory Me²⁺ and of Ca²⁺ on basal and hormone-stimulated cyclic nucleotide levels were studied in the ductus deferens of the rat.In tissue segments that had been preincubated with no Me²⁺ added, Mg²⁺ (2 or 10 mM), Co²⁺, Ni²⁺ and Cd²⁺ (2 mM each) caused 2-fold elevations of the cyclic GMP level within 5 min, whereas Mn²⁺ and Ca²⁺ caused 7-and 5-fold elevations, respectively. Cyclic AMP levels were not significantly changed except for a 10–20% increase by Mn²⁺. Whereas Mn²⁺ caused a continuous rise of the cyclic GMP level over at least 10 min, the stimulatory effect of Ca²⁺ was most pronounced during the first minute. The effects of Ca²⁺ and Mn²⁺ were not additive. When Ca²⁺ was added with or after Mn²⁺ (2 mM each), the stimulatory effect of Mn²⁺ was depressed. Similarly, Mn²⁺ caused only a small increase in the cyclic GMP level of tissue incubated with Ca²⁺.With the hormonal stimuli, acetylcholine and noradrenaline (0.1 mM each), a dissociation of their effects on tissue tone and on the cyclic GMP level occurred. Whereas either stimulus caused about 2-fold elevation of the cyclic GMP in the presence of Mn²⁺ with or without Ca²⁺ present, contractile responses were prevented by Mn²⁺. In contrast, K⁺ at a high, depolarizing concentration, which increased the cyclic GMP level in the presence of Ca²⁺, had no effect on the cyclic nucleotide with Mn²⁺ added instead of Ca²⁺. Whereas hormone-induced elevations of the cyclic GMP level depended on the presence of Me²⁺, the effect of hydroxylamine (0.1 mM) to increase the level of this nucleotide in the absence of Me²⁺ was not augmented by Ca²⁺ or Mn²⁺.The present data indicate that hormone-induced elevations of the cyclic GMP level in the absence of extracellular Ca²⁺ do not promote contraction. The findings also suggest that hormones are capable of stimulating cyclic GMP formation by mechanisms other than hormone-induced increase in cytoplasmic Ca²⁺ concentration.Abbreviations cGMP cyclic - GMP guanosine 3:5-monophosphate - cAMP cyclic AMP, adenosine 3:5-monophosphate - Me²⁺ divalent cation(s) - EDTA ethylenediaminetetraacetic acid This work was supported by the Deutsche Forschungsgemeinschaft. A preliminary report was presented (Schultz and Schultz, 1976). The data have partially been taken from a dissertation submitted by K.D.S. to the University of Heidelberg in partial fulfillment of the requirements for the Ph. D. degree     

Exposure of rats to high concentrations of 1,1,1-trichloroethane and its effects on brain lipid and fatty acid composition

Exposure of rats to 1,1,1-trichloroethane (TRI) (1200 p.p.m.) for 30 days resulted in changes in the fatty acid pattern of the brain ethanolamine phosphoglyceride. A decrease was observed in stearic acid (18:0) and arachidonic acid (20:4), while the 22-carbon (n-3) fatty acids were increased. These changes in the fatty acid pattern were similar to that observed previously in the rat for another solvent, perchloroethylene, at a lower exposure concentration (320 p.p.m). Both these solvents are little metabolised and it seems that a common mechanism exists whereby these solvents alter the fatty acid pattern of brain phospholipid upon exposure. The relatively low uptake of TRI makes a high exposure level (1200 p.p.m.) necessary to attain a blood concentration high enough for the changes to appear.     

Effects of 1,1,1-trichloroethane administered by different routes and in different solvents on barbiturate hypnosis and metabolism in mice.

A 24-hr inhalation of 1,1,1-tricholoroethane (methylchloroform), 3,000 ppm, reduced pentobarbital hypnosis and increased hexobarbitaloxidation by the 9,000 x g liver supernatant fraction in male mice. On the other hand, an ip injection of methylchloroform, l ml/kg, increased the duration of pentobarbital hypnosis and reduced hexobarbital metabolism by the liver microsomal enzymes. The potentioating effect of methylchloroform on pentobarbital hypnosis as diminished when it was diluted with olive oil, but was markedly enhanced when diluted with dimethysusoxide (DMSO) before injection. Three local applications of methylchloroform )1:1 dilution with DMSO) had an effect similar to that of inhalation on pentobarbital hypnosis.     

Effects of atriopeptins on relaxation and cyclic GMP levels in rat and rabbit aortas

The effects of atriopeptins I and II on relaxation and cyclic GMP levels were studied on rat and rabbit aortas. Atriopeptin I was 2- and 100-fold less potent than atriopeptin II in causing relaxation of rat and rabbit aortas, respectively. The atriopeptin-elevated cyclic GMP levels generally correlated with the amount of relaxation. These results demonstrate that the vasodilator profile and, presumably, the receptor for atrial natriuretic factor, varies among different blood vessels and species.     

The uptake and elimination of 1,1,1-trichloroethane during and following inhalation exposures in rats

The pharmacokinetics of 1,1,1-trichloroethane (TRI) was studied in male Sprague-Dawley rats in order to characterize and quantify TRI uptake and elimination oby direct measurements of the inhaled and exhaled compound. Fifty or 500 ppm TRI was inhaled for 2 hr through a one-way breathing valve by unanesthetized rats of 325-375 g. Repetitive samples of the separate inhaled and exhaled breath streams, as well as arterial blood, were collected concurrently both during and following TRI inhalation and analyzed for TRI by gas chromatography. Respiratory rates and volumes were continuously monitored during and following exposure and were used in conjunction with the pharmacokinetic data to characterize profiles of uptake and elimination. TRI was very rapidly absorbed from the lung, in that substantial levels were present in arterial blood at the first sampling time (i.e., 2 min). Blood and exhaled breath concentrations of TRI increased rapidly after the initiation of exposure, approaching but not reaching steady state during the 2-hr exposures. The blood and exhaled breath concentrations were directly proportional to the exposure concentration during the exposures. Percentage uptake of TRI decreased 30-35% during the first hour of inhalation, diminishing to approximately 45-50% by the end of the exposure. Total cumulative uptake in the 50 and 500 ppm groups over the 2-hr inhalation exposures was determined to be 6 and 48 mg/kg body wt, respectively. By the end of the exposure period, 2.1 and 20.8 mg, respectively, of inhaled TRI was eliminated from rats inhaling 50 and 500 ppm TRI. A physiological pharmacokinetic model for TRI inhalation was utilized to predict blood and exhaled breath concentrations for comparison with observed experimental values. Overall, values predicted by the physiological pharmacokinetic model for TRI levels in the blood and exhaled breath were in close agreement with measured values both during and following TRI inhalation.     

Evaluation of 1,1,1-trichloroethane and flurothyl locomotor effects following diazepam treatment in mice.

The abused volatile solvent 1,1,1-trichloroethane (TCE) shares many acute behavioral effects with central nervous system (CNS) depressants; however, demonstration of tolerance to these effects has been difficult. The purpose of the present study was to investigate the development of TCE-induced changes in locomotor activity in mice following repeated injections with diazepam. In the initial concentration-effect curve determinations, diazepam decreased locomotor activity at all doses tested and TCE produced a biphasic effect, increasing locomotor activity at lower concentrations with return to control levels at a high (16,000 ppm) concentration. Flurothyl, a vapor with convulsive properties, had no pronounced effects on locomotor activity at subconvulsant concentrations. Following four daily injections with vehicle or with 10 mg/kg/day diazepam, mice were administered the same concentration of drug/inhalant that they received initially and were retested for locomotor activity effects. Concentration-effect curves for diazepam and flurothyl were not altered by this modest regimen of repeated dosing with diazepam. In contrast, sensitization to the locomotor-stimulating effects of TCE was observed in diazepam-treated mice, but not in vehicle-treated mice. These results suggest that the development of sensitization to TCE involves common mechanisms with those that are affected by repeated dosing with the CNS depressant diazepam.     

[14C]Methyl chloroform (1,1,1-trichloroethane): Pharmacokinetics in rats and mice following inhalation exposure

Studies on the pharmacokinetics of [¹⁴C]methyl chloroform (1,1,1-trichloroethane) in male Fischer 344 rats and B6C3F1 mice were undertaken to characterize the disposition of the inhaled chemical over a wide range of exposure concentrations. The animals were exposed to 150 or 1500 ppm of [¹⁴C]methyl chloroform vapor for 6 hr and the elimination of ¹⁴C activity was followed for 72 hr. Following exposure to either concentration of methyl chloroform, both species excreted >96% of the total recovered radioactivity during the first 24 hr. The major route of elimination of methyl chloroform was via exhalation of unchanged chemical in the expired air which constituted approximately 94–98% of the total recovered radioactivity in rats and 87–97% in mice at 150 and 1500 ppm, respectively. Mice were found to eliminate methyl chloroform in the expired air more rapidly than did rats. The remaining radioactivity (approximately, 2–13%) was detected as metabolized methyl chloroform in the expired air (¹⁴CO₂) and as nonvolatile radioactivity in the urine, feces, carcass, and cage wash. Although mice were found to metabolize two to three times more methyl chloroform on a body weight basis, the biotransformation of methyl chloroform was shown to be a saturable, dose-dependent process in both species. Since the biotransformation of methyl chloroform occurred to such a limited extent, saturation of its metabolism did not impact markedly on the distribution or elimination of the parent chemical. The body burden, end-exposure blood level, and tissue concentration of methyl chloroform were found overall to increase in direct proportion with the exposure concentration. [¹⁴C]Methyl chloroform was more concentrated in the fat of both species than in the liver or kidneys immediately after exposure. However it was rapidly cleared from the fat so that by 24 hr <2% of the initial radioactivity remained. Thus, methyl chloroform shows little potential for significant bioaccumulation in rodents.     

Trichloroethylene and 1,1,1-trichloroethane: effects on brain and liver after five days intermittent inhalation

The inhalation exposure of adult male rats to 7.9 mol/l (200 ppm) of trichloroethylene for 4 days 6 h each day led to an accumulation of the solvent in the perirenal fat 17 h after the last exposure. Exposures to 20 mol/l (500 ppm) of 1,1,1-trichloroethane caused similar accumulation. The latter rats presented no changes in their behaviour in an open-field test whereas the motor behaviour of the animals exposed to trichloroethylene was more active in comparison to controls 1 h after the exposure on the 4th day. Trace effects of trichloroethylene on emotional behaviour of the same rats could be seen 17 h after the last exposure.Further exposures on the 5th day increased brain, liver, lung and blood contents of trichloroethylene and 1,1,1-trichloroethane. A slight decrease in brain RNA content was found in the 1,1,1-trichloroethane exposed rats while RNA decreased significantly after the fifth day of trichloroethylene inhalation. The exposure to 1,1,1-trichloroethane on the 5th day depressed also the microsomal cytochrome P-450 content in liver of rats whereas trichloroethylene increased the hemochrome content slightly at the same time.     

Acute, short-term, and subchronic oral toxicity of 1,1,1-trichloroethane in rats.

1,1,1-Trichloroethane (TRI) is a widely used solvent that has become a frequent contaminant of drinking water supplies in the U.S. There is very little information available on the potential for oral TRI to damage the liver or to alter its P450 metabolic capacity. Thus, a major objective of this investigation was to assess the acute, short-term, and subchronic hepatotoxicity of oral TRI. In the acute study, male Sprague-Dawley (S-D) rats were gavaged with 0, 0.5, 1, 2, or 4 g TRI/kg bw and killed 24 h later. No acute effects were apparent other than CNS depression. Other male S-D rats received 0, 0.5, 5, or 10 g TRI/kg po once daily for 5 consecutive days, rested for 2 days, and were dosed for 4 additional days. Groups of the animals were sacrificed for evaluation of hepatotoxicity 1, 5, and 12 days after initiation of the short-term experiment. This dosage regimen caused numerous fatalities at 5 and 10 g/kg, but no increases in serum enzymes or histopathological changes in the liver. For the subchronic study, male S-D rats were gavaged 5 times weekly with 0, 0.5, 2.5, or 5.0 g TRI/kg for 50 days. The 0 and 0.5 g/kg groups were dosed for 13 weeks. A substantial number of rats receiving 2.5 and 5.0 g/kg died, apparently due to effects of repeated, protracted CNS depression. There was evidence of slight hepatocytotoxicity at 10 g/kg, but no progression of injury nor appearance of adverse effects were seen during acute or short-term exposure. Ingestion of 0.5 g/kg over 13 weeks did not cause apparent CNS depression, body or organ weight changes, clinical chemistry abnormalities, histopathological changes in the liver, or fatalities. Additional experiments did reveal that 0.5 g/kg and higher doses induced hepatic microsomal cytochrome P450IIE1 (CYP2E1) in a dose- and time-dependent manner. Induction of CYP2E1 activity occurred sooner, but was of shorter duration than CYP2B1/2 induction. CYP1A1 activity was not enhanced. In summary, 0.5 g/kg po was the acute, short-term, and subchronic NOAEL for TRI, for effects other than transient CYP2E1 induction, under the conditions of this investigation. Oral TRI appears to have very limited capacity to induce P450s or to cause liver injury in male S-D rats, even when administered repeatedly by gavage in near-lethal or lethal dosages under conditions intended to maximize hepatic effects.     

Effects of atriopeptins on relaxation and cyclic GMP levels in human coronary artery in vitro

The effects of atriopeptins on relaxation and cyclic GMP levels were examined in human coronary artery. alpha-Atrial natriuretic polypeptide and atriopeptins I, II and III all induced relaxation. Relaxations to atriopeptin I were of a smaller magnitude. The atriopeptins elevated cyclic GMP levels from 2- to 3-fold. These studies suggest that atriopeptins released from the heart may dilate the vasculature of this organ and increase coronary blood flow through the formation of cyclic GMP.