Combined effects of paired solvents on the rat's auditory system.

A number of volatile organic solvents have been shown to be ototoxic to rats, but there is little information regarding how solvents might act in this way when encountered in combination. To examine this issue, male Long Evans rats were exposed by inhalation to pairs of solvents known to be ototoxic when administered individually; those reported on here are trichloroethylene+toluene, mixed xylenes+trichloroethylene, xylenes+chlorobenzene, and chlorobenzene+toluene. Rats were exposed 8 h/day for 5 consecutive days, using complementary proportions of isoeffective concentrations of the solvents alone. Hearing was assessed by brainstem-evoked response audiometry. The effects were as predicted by a linear dose-addition model, indicating additive rather than synergistic or antagonistic interactions at the concentrations studied.

Solvent-induced ototoxicity in rats: an atypical selective mid-frequency hearing deficit.

Most previous reports of ototoxicity following exposure to several volatile organic solvents have restricted testing to the low- and mid-frequencies (2-20 kHz) of the hearing range in the rat (0.25-80 kHz). We report here that inhalation exposure to styrene, mixed xylene, toluene, and 1,1,2-trichloroethylene resulted in hearing dysfunction only in the mid-frequency range and spared function at lower and higher frequencies. Adult male Long Evans rats were exposed via inhalation (whole body) in flow-through chambers. The following exposures were used: styrene, 1600 ppm; 1,1,2-trichloroethylene, 3500 ppm; toluene, 2500 ppm; mixed xylenes, 1800 ppm (N = 7-8 per group, 8 h/day for 5 days), and n-butanol, 4000 ppm (N = 10/group, 6 h/day for 5 days). Testing of auditory function was conducted 5 to 8 weeks after exposure using reflex modification audiometry (RMA). RMA thresholds were determined for frequencies from 0.5 to 40 kHz. Results indicated increased RMA thresholds for the mid-frequency tones (e.g., 8 and 16 kHz), but not higher or lower tones, for all solvents except n-butanol. Toluene and xylene also increased thresholds at 24 kHz. These data indicate that for those solvents reported thus far to cause hearing loss, the deficit is restricted to mid-frequencies in rats.

The neuroepidemiology of styrene: a critical review of representative literature.

Because exposure to styrene occurs commonly in some industries and styrene is highly lipid soluble, it is reasonable to be concerned about the possibility that styrene is neurotoxic. Styrene, like many other solvents, volatile anesthetics, and drugs, does, at certain concentrations, produce acute changes in consciousness with consequent alterations of feelings, cognition, and psychomotor functioning. Such acute actions do not imply that styrene also would produce reversible or irreversible damage to the nervous system; the evaluation of long-term exposures to styrene also is necessary to draw conclusions about the full range of neural effects that styrene might produce. To that end, several studies of workers exposed to styrene for up to 30 years have been undertaken in factories in many parts of the world. Epidemiologists have suggested that neuropsychological deficits such as slowing of reaction time, loss of color vision, and vestibulooculomotor dysfunction are reliably induced by styrene at levels near or below current exposure standards, which range from 20 to 50 ppm in most of the world. However, the workers so studied always were described as healthy, and the effects noted were considered to be subclinical. A detailed evaluation of much of the neuroepidemiological literature on styrene (38 papers and related literature), however, indicated that the findings were, almost universally, false positive outcomes due to (1) type I statistical error, (2) the action of some factor other than styrene, and (3) misinterpretation of data. Despite the study of workers exposed for many years, no indications of persisting damage to the nervous system were evident from this review. The conclusions of this review of the neuroepidemiology of styrene are consistent with those based on critical reviews of the solvent literature in general, with specific reference to the probable absence of such an entity as the "painter's syndrome" or "chronic toxic encephalopathy". Because the results on styrene neurotoxicity that provide an inclination to lower the current threshold limit values (TLVs) are false positive findings, there is no scientific basis for a reduction in the current TLV.

Combined effects of solvents on the rat's auditory system: styrene and trichloroethylene.

Because exposures to toxic agents typically involve more than one substance, it is necessary to know if combined exposures pose different risks than those to single agents. Many solvents have been implicated in central nervous disorders and some of them are known to produce hearing loss, probably mediated by damage to cochlear hair cells. Hearing loss was studied by recording the brainstem auditory evoked response (BAER) in male Long Evans rats exposed 8 h/day for 5 days to mixtures of styrene (STY) and trichloroethylene (TCE). Dose groups included air or solvent pairs (STY/TCE) in the following concentrations (ppm): (0:3000), (250:2250), (500:1500), (750:750) and (1000:0). Decreased BAER amplitude, indicative of hearing loss, was correlated with blood levels of total solvent. The effects were as predicted by a linear dose-addition model, indicating neither synergistic nor antagonistic interactions at the concentrations studied.

Rare events and the CNV--the oddball CNV.

Event-related brain potentials (ERPs), such as Nd and P300, change as a function of the proportion of time if a rare, significant, stimulus occurs. This 'oddball' paradigm has had a significant influence on the interpretation of ERPs in terms of the psychological and information-processing functions they reflect. Interpretations of transient components have differed considerably from that of the sustained potential known as the Contingent negative Variation (CNV), the latter appearing to reflect proactive rather than reactive brain processes. However, experiments in monkeys indicated that the CNV was also sensitive to the oddball effect. The purpose of this experiment was to replicate that finding in humans. The CNVs and P300s of five male and five female college students were studied under three conditions involving different proportions of two types of warning stimuli in a cued reaction time task (paired tone and light). The proportions of one of the warning stimuli, with respect to the total number of trials, were 0.10, 0.30 and 0.50. P300s were larger when elicited by rare warning tones only in the 0.10 condition with maximum P300 amplitude at the parietal site. CNVs at frontal and central areas were larger when the warning cue was the rare event, but there was no effect at the parietal site. These findings may require a rethinking of specific information processing interpretations of other endogenous ERPs, although the results also indicate that the 'oddball' effect on the P300 and CNV was distinctive in terms of scalp distributions and sensitivity to the manipulation.

Interactive effects of toluene and hexane on behavior and neurophysiologic responses in Fischer-344 rats.

Solvent mixtures are ubiquitous in industrialized environments and are used frequently for recreational purposes. Toluene and hexane are common components of many solvent mixtures and have characteristic, but different, neurotoxic consequences. Interestingly, Takeuchi et al., (1981) reported that toluene attenuated the peripheral neuropathy caused by n-hexane, possibly by blocking its metabolism to 2,5-hexanedione (Perbellini, et al., 1982). To confirm such effects at higher concentrations and to examine effects on the central nervous system (CNS), four groups of 12 rats each were exposed to air, toluene (1200 ppm), hexane (4000 ppm), or a mixture of toluene (1200 ppm) and hexane (4,000 ppm) 14 hr/day for 9 weeks. A battery of behavioral and electrophysiologic tests was used to assess the functional consequences of their exposures. The battery consisted of measures of grip strength, locomotor gait and landing splay, sensory sensitivities during conditioned avoidance performance, the action potential of the ventral caudal nerve, and the brainstem auditory evoked response. Measures of peripheral nervous system functions (e.g., grip strength and conduction velocity) showed interactive effects like those reported by Takeuchi et al. Toluene greatly reduced the neuropathy caused by hexane. Hexane-induced abnormalities in central components of the brainstem response were much less reduced in the presence of toluene. There was no reciprocal action of hexane on the motor syndrome and hearing loss caused by toluene.

A procedure for using proton magnetic resonance imaging to determine stereotaxic coordinates of the monkey's brain.

Because of great inter-individual variability in the sizes and configurations of monkey brains, consistent intracerebral stereotaxic placements are not possible when a general brain atlas is relied upon to derive placement coordinates. We describe a procedure that allows the determination of brain atlases for individual monkeys using proton magnetic resonance imaging and the translation of image coordinates to stereotaxic coordinates. The latter is accomplished by chronically implanting glass beads filled with copper sulfate into the skull to establish a plane horizontal to the stereotaxic plane and to provide reference points for zeroing stereotaxic carriers during intracerebral implants. The efficacy of this procedure was confirmed experimentally.

The hearing loss associated with exposure to toluene is not caused by a metabolite.

Exposure to toluene causes a marked hearing loss in rats, and this effect has been observed in some human solvent abusers. The issue of whether toluene or one of its metabolites is responsible for this effect has not been examined. To attempt to resolve this issue, we manipulated the metabolism, and thus the circulating levels, of toluene as follows. Two groups of rats were exposed to phenobarbital (PB) in their drinking water (0.1%) for seven days to induce detoxifying liver enzymes; two other groups had access to PB-free water. Then half of the rats exposed to PB or water were exposed to filtered air or a concentration of toluene expected to cause hearing loss. Levels of toluene in blood were markedly reduced by the PB and the excretion of hippuric acid was increased. All rats were tested for auditory sensitivity by brainstem auditory-evoked response (BAER) audiometry using a 16-kHz tone pip. The rats exposed to toluene alone showed a marked reduction in the integrated BAER waveform, indicative of the expected hearing deficit. None of the other treated rats showed any deviation from controls (i.e., water and air). These results provide strong evidence that toluene itself is responsible for the auditory dysfunction. Toluene also caused the rats to increase their fluid consumption and urine output; these effects were not altered by PB. Identification of toluene as the proximal ototoxicant should facilitate the search for the mechanism of this effect.

Sensory-evoked potentials in rats chronically exposed to trichloroethylene: predominant auditory dysfunction.

Sensory-evoked potentials (EPs) were studied in male Long-Evans and Fischer-344 rats in order to characterize the electrophysiological consequences of chronic inhalation exposure to trichloroethylene (TCE). Groups of ten Long-Evans rats were exposed to air or 1600 ppm or 3200 ppm TCE for twelve weeks and evaluated periodically with a multisensory test battery. Brainstem auditory-evoked response (BAER) amplitudes were depressed by TCE, whereas somatosensory and visual potentials remained normal. The effects on BAERs, which varied with tone intensity and frequency, suggested that TCE causes a predominantly high-frequency hearing loss. Comparable effects were obtained in both strains of rats and were like those previously observed following exposure to toluene.

Preliminary study on subcortical slow potentials related to the readiness potential in monkey.

Two female rhesus monkeys with cortical and subcortical DC-electrodes were studied during self-paced operant responding to determine the feasibility of mapping the intracerebral distribution of readiness potentials (RP). These potentials reflect preparatory aspects of motor activation and may be useful indicators of subcortical areas involved in preparatory set. The monkeys had learned to execute the task appropriately after 17 and 28 sessions respectively; one monkey's performance was very stable, and the other's erratic. However, relatively comparable RPs were recorded from the monkeys when recordings from sessions involving good performance were considered. RPs of relatively large amplitude appeared in electrodes positioned near the substantia nigra and pretectal/collicular region; smaller responses were observed in cortex and midbrain reticulum and, in one monkey, the caudate nucleus. These findings indicate the feasibility of such studies and suggest that, like the contingent negative variation, RPs occur in many subcortical regions.

Hippocampal damage associated with prolonged glucocorticoid exposure in primates.

In the laboratory rat and guinea pig, glucocorticoids (GCs), the adrenal steroids that are secreted during stress, can damage the hippocampus and exacerbate the hippocampal damage induced by various neurological insults. An open question is whether GCs have similar deleterious effects in the primate hippocampus. In fact, we showed that sustained and fatal stress was associated with preferential hippocampal damage in the vervet monkey; however, it was not possible to determine whether the excessive GC secretion that accompanied such stress was the damaging agent. The present study examines this possibility. Pellets of cortisol (the principal GC of primates) were stereotaxically implanted into hippocampi of 4 vervet monkeys; contralateral hippocampi were implanted with cholesterol pellets as a control. One year later at postmortem, preferential damage occurred in the cortisol-implanted side. In the cholesterol side, mild cell layer irregularity was noted in 2 of 4 cases. By contrast in the cortisol-exposed hippocampi, all cases had at least 2 of the following neuropathologic markers: cell layer irregularity, dendritic atrophy, soma shrinkage and condensation, or nuclear pyknosis. Damage was severe in some cases, and was restricted to the CA3/CA2 cellfield. This anatomical distribution of damage, and the cellular features of the damage agree with that observed in instances of GC-induced toxicity in the rodent hippocampus, and of stress-induced toxicity in the primate hippocampus. These observations suggest that sustained GC exposure (whether due to stress, Cushings syndrome or exogenous administration) might damage the human hippocampus.

Acute interactive pharmacologic effects of inhaled toluene and dichloromethane on rat brain electrophysiology.

Toluene (TOL) and dichloromethane (DCM) are widely used industrial solvents and are common components of solvent mixtures that are voluntarily inhaled to produce altered states of consciousness. In previous studies we characterized some of the acute electrophysiologic effects of these solvents. Opposite effects were noted for some measures, suggesting that they might be antagonistic when combined. In this study we examined the solvents again singly (10,700 and 16,000 ppm) and also in combination (16,000 ppm: 33/67 and 67/33% TOL/DCM ratios). The single gases caused effects similar to those observed previously. Combined effects varied, dependent upon the particular variable examined and the major gas in the mixture. In some respects the solvents were concordant, exerting similar effects on a variable, e.g., both solvents prolonged the latencies of components of the brainstem auditory-evoked response. In other respects they were discordant, e.g., whereas toluene caused mean EEG frequency to increase, dichloromethane had the opposite effect. Sometimes the solvents had similar effects alone, but acted independently in combination. Nonindependent interactions were also observed-both additive/subtractive or positively or negatively synergistic. The results further demonstrate and emphasize the unique patterns of acute central nervous system effects that can be effected by solvents that might have a common behavioral endpoint such as anesthesia, and the results characterize a variety of electrophysiologic interactions between these two solvents. Although there were several variables exhibiting synergistic relationships, independent or additive interactions were the most common.

Acute effects of inhaled dichloromethane on the EEG and sensory-evoked potentials of Fischer-344 rats.

Acute effects of inhaled dichloromethane on the spontaneous electroencephalogram (EEG) and sensory-evoked potentials (EPs) were characterized and compared to previously observed effects of toluene; both solvents are common components of abused solvent mixtures. Twelve adult male Fischer-344 rats with chronic epidural electrode implants served as subjects. Each rat was exposed for 60 min to 5,000, 10,000, and 15,000 ppm dichloromethane while held in a plastic restrainer that also served as a head-only exposure chamber. The sequence of exposures was counterbalanced across rats, and the exposures were separated by about one week. To characterize the time course of any changes, somatosensory and flash EPs were recorded every 5 min during the first 45 min of the exposures. As was the case with toluene, electrophysiologic waveforms recorded from different sensory systems, and components of these waveforms, reacted in different ways to dichloromethane. With respect to the FEP and SEP the two solvents produced quite different effects. Toluene increased the amplitudes of early FEP components, eliminated late components, induced oscillations in visual cortex, and had no discernible effects on component latencies. In contrast, dichloromethane eliminated the N1 component, at moderate exposure had little or no effects on amplitudes of the later components (N3 through N4), did not induce oscillations, and had significant effects on latencies. Whereas toluene dramatically increased SEP component amplitudes at moderate concentrations with diminishing effect at higher concentrations and exposure times, dichloromethane rather uniformly decreased SEP amplitude in a simple concentration-related way. Toluene and dichloromethane had similar effects on BAER component latencies. They both caused component (P1 through P5) latencies and the P1-P5 interwave time to increase. However, whereas toluene increased early and late (but not middle) component amplitudes, dichloromethane decreased the amplitudes of early and late components and increased the amplitudes of middle components. These results emphasize the acute pharmacologic specificity of different solvents and suggest that differences in chronic neurotoxicity might also be found; they also suggest that predictable interactions might be found with acute and chronic exposure to mixtures that contain such solvents.

Effect of acute respiratory acidosis on multimodality sensory evoked potentials of Long-Evans rats.

Auditory, visual and somatosensory evoked potentials (EPs), recorded epidurally from 31 chronically implanted male Long-Evans rats, were studied to examine the pattern of sensory effects caused by hypercapnia. Recordings were obtained before exposures, 10-20 min after the beginning of exposure to CO2 in synthetic air, and 30 min after the end of exposure. Previous recordings revealed no substantial effects of the extended recording period itself. Blood pH during an average exposure of 18.8% CO2 was about 7.1. During this level of CO2 exposure the somatosensory response was almost completely abolished, but the latencies of early detectable components were not affected. In contrast, the latencies of all brainstem auditory evoked response components and the 1-5 interwave time increased, whereas amplitudes were only slightly affected. Amplitudes and latencies of early and late components of the flash EP were decreased and lengthened, but the after-discharge components appeared to be most sensitive to CO2. Concentration-response relationships were examined by exposure of rats to 8 and 16% CO2. The most sensitive EP parameter was average amplitude of the late somatosensory EP components. These results suggest that EPs might be useful for assessing acute metabolic disturbances as well as more commonly assessed neurologic disorders.

A method for exposing primates to marihuana smoke that stimulates the method used by human marihuana smokers.

A method was developed for exposing rhesus monkeys to marihuana smoke under conditions simulating those used by human marihuana smokers. An ADL II smoking machine was used to generate puffs of smoke of constant volume from marihuana or placebo cigarettes at a constant rate. The outlet of the smoking machine was connected by Tygon tubing to an airtight face mask covering the monkey's nose and mouth. The monkey was seated in an airtight Plexiglas chamber with only its head protruding. When a puff of smoke was generated, a vacuum was imposed on the chamber causing the monkey to inhale deeply and hold its breath. After 6 seconds, the vacuum was removed and the monkey was allowed to exhale and breathe fresh air freely until the next puff. Delivery of smoke to the monkey by this method was more efficient than allowing the monkey to voluntarily inhale the smoke. The method was used in acute dose-effect experiments and, subsequently, in long-term experiments. It may be useful with other animal species and for efficiently delivering other airborne substances of abuse.

Acute electrophysiologic effects of inhaled toluene on adult male Long-Evans rats.

Experiments were carried out in Long-Evans rats to verify and extend previous findings about the effects of toluene on sensory-evoked potentials (EPs) of Fischer-344 rats. Inhalation exposures to 3000 and 8000 ppm in Long-Evans rats confirmed that toluene 1) transiently enhances certain components of somatosensory, flash- and click-evoked (brainstem) potentials, 2) increases the latencies and interwave times of brainstem auditory-evoked responses, 3) depresses late components of the flash EP, 4) induces high frequency oscillations in the visual cortex, and 5) produces both facilitatory and suppressant effects on EPs, dependent on exposure concentration and time. New results indicated that toluene 1) has similar effects on Long-Evans as it does on Fischer-344 rats, 2) increases EEG theta activity, 3) has minor effects on cortical auditory and pattern-reversal EPs (PREP), but suppresses the steady-state PREP, and 4) induces oscillations in the visual cortex, irrespective of the presence of flashes.

Multimodal effects of acute exposure to toluene evidenced by sensory-evoked potentials from Fischer-344 rats.

Male Fischer-344 rats were exposed by inhalation to 500, 2000, 5000, 8000 and 16000 ppm toluene for 30 min in two experiments. Exposures up to 8000 ppm in Experiment 1 caused concentration-related changes in the click-elicited brainstem auditory-evoked response (CBAER), flash-evoked potential (FEP) and somatosensory-evoked potential (SEP). Latencies of CBAER components were prolonged and amplitudes of late components were increased by toluene. Toluene did not detectably alter the latencies of FEP or SEP components. Early FEP component-amplitudes were increased and late component-amplitudes were decreased; toluene also induced a poststimulus oscillation in the FEP. Most component-amplitudes of the SEP were substantially increased, but N2P2 amplitude appeared to be more sensitive than other components to depressant effects of the solvent. The same effects on the CBAER were observed in Experiment 2, but a more substantial increase in the amplitudes of late components elicited by tone pips suggested that frequency-dependent cochlear irritation might underlie previously observed subchronic ototoxicity. These effects were increased by exposure to 16000 ppm toluene. Effects like those observed in Experiment 1 were noted on the FEP, but the oscillations were less with exposure to 16000 than 8000 ppm. Changes in the SEP were evident within 2 minutes of exposure onset, and amplitudes increased over the course of about 15 min, leveling off or decreasing thereafter. The amplitude of the N2P2 component was again less influenced than other components during exposure to 8000 ppm and was reduced to less than baseline amplitude by 16000 ppm. Effects of concentration and rates of development and recovery were systematically related to SEP component latency. Toluene appears to have both enhancing and inhibiting effects on neural pathways serving sensory systems, depending on the modality and the site of generation of the components within modalities. A particular balance between these properties might relate to the hedonic characteristics of this abused solvent.