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

Pharmacology BiochemistO" & Behm'ior. Vol. 34, pp. 619-629. o PergamonPress plc, 1989. Printedin the U.S.A.

0091-3057/89 $3.00 + .00

Acute Effects of Inhaled Dichloromethane on the EEG and Sensory-Evoked Potentials of Fischer-344 Rats C H A R L E S S. R E B E R T , M I C H A E L J. M A T T E U C C I A N D G O R D O N T. P R Y O R

SRI International, LAll7,

333 Ravenswood Ave., Menlo Park, CA 94025

R e c e i v e d 16 F e b r u a r y 1989

REBERT, C. S., M. J. MATTEUCCI AND G. T. PRYOR. Acute effects of inhaled dichloromethane on the EEG and sensoo'-evoked potentials of Fischer-344 rats. PHARMACOL BIOCHEM BEHAV 34(3) 619-629, 1989.--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 rain 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, dichlorometbane 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 PI-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. Dichloromethane

Solvent abuse

EEG

Sensory-evoked potentials

DURING the last decade there has been increasing concern about neurotoxic sequelae of chemicals encountered in the environment, those self-administered for their psychoactive properties, and agents administered for therapeutic reasons (30). This concern has been accompanied by the search for sensitive and reliable methods for assessing neurotoxicity (2, 12, 16). One area of particular concern, because of high doses or concentrations of substances administered, is that of substance abuse. In this context the abuse of volatile solvents is of special concern because of the very young age of most abusers and the paucity of data concerning the effects of various solvents on the nervous system and their relative dangers (33). Some components of abused solvent mixtures are unquestionably neurotoxic (e.g., the hexacarbons), whereas the neurotoxicities of other solvents are less obvious, e.g., toluene [although toluene is known to cause irreversible hearing loss in rats (20)].

Rats

Because of their psychoactive properties, the acute neurobehavioral effects of solvents have also been of interest in the context of substance abuse and worker safety (7,33). Knowing something about the profile of central nervous system (CNS) effects of abused substances might provide clues about why they are abused and what kinds of chronic effects might be expected; such information may also suggest specific areas of acute dysfunction that might occur with environmental exposures. Of the variety of ways CNS functions can be assessed, electroencephalographie (EEG) and evoked potential (EP) recordings provide a bridge between more direct neuronal recording and behavioral observations (8, 14, 24). Widespread use of these techniques in neurology led to their extrapolation to neurotoxicology. It is only recently, however, that the analysis of acute effects of solvents on the CNS have been evaluated by EPs. Of particular interest here are recent findings on toluene, which have shown that

~Copies of unpublished reports cited may be obtained from the authors.

619

620

REBERT, MATTEUCCI A N D PRYOR

EPs obtained in different sensory modalities are not equally affected by that solvent and that different waveform components within modalifies are also not always affected the same (10, 15, 28, 29). It is also clear that different solvents have different profiles of acute behavioral effects (23), and one would expect a similar outcome with respect to electrophysiologic measures. However, there have been very few such studies. Therefore, we are examining the effects of several solvents on parameters of an EEG/EP battery of tests (24, 28, 29). Dichloromethane (DCM) is a common component of solvent mixtures, including several that are subject to abuse. It is a widely used industrial solvent and was used as an anesthetic for many years (37). From the latter, it obviously has acute neurobehavioral effects; it has been shown to affect nonspecific motor activity, learning, vestibulo-ocular function, auditory vigilance, critical flicker frequency, and sensory and cognitive functions (t, 7. 36, 37). Pankow et al. (18) demonstrated a slowing of sciatic motor conduction velocity in rats, and Stewart et al. (35) showed that DCM reduces the amplitude of visual EPs in humans. Persistent sequelae of exposures to DCM have been less well documented (37). However, because one of its metabolic byproducts is carbon monoxide (CO), DCM may contribute to long-term deleterious effects in three ways--lowering the threshold for metabolic compromise because of its CO metabolite, through the consequences of nonspecific narcotic actions, and, as a component of mixtures, via interactions with other substances (3). The aims of the present investigation were 1) to determine if DCM would affect EPs in rats and, if so, whether the effects would differ for different modalities of stimulation; 2) to study the effects of various concentrations of DCM on EPs; 3) to examine the time course of any changes during acute exposures; and 4) to compare the effects of DCM with those obtained earlier with toluene. Rats were used in this study because of their obvious general utility in toxicologic investigations, and to provide data directly comparable to earlier studies of toluene using the Fischer-344 strain. The exposure levels used were high with respect to threshold limit values established for human industrial exposures (175 ppm for short-term exposure), but were relevant to the high concentrations employed by individuals who inhale solvents to induce altered states of consciousness. For some measures of EPs in rats our results suggest that effects would be observed at concentrations below the lowest used in this investigation (5,000 ppm). Since changes in visual EPs in humans have been reported at concentrations of 500 to 1,000 ppm (35), it is likely that the sensitivity of the rat and human to the acute neurologic effects of DCM are quite similar. METHOD

Subjects and Surgical Preparation Twelve male Fischer-344 rats, with average weight at the beginning of the experiment of 274 g, were used as subjects. They were kept in 23 × 1 4 × 4 5 cm pIastic cages (3/cage) housed in laminar flow racks. Food and water were available ad lib, Lights were on from 7 a.m. to 7 p.m. Surgical procedures were like those described before (24,26). Epidural stainless steel bolts (0-80 × 1/8 in.) were placed in the cranium over visual (6.0 mm posterior to bregma and 3.5 mm to the right of the midline) and somatosensory (2.0 mm posterior to bregma and 2,0 mm left of midline) cortices and in midline frontal bone over the olfactory bulbs (reference electrode). Wires were soldered to the bolts prior to implant to preclude heat damage to the cortex (11). Hooks embedded in the acrylic headplug were used during testing to hold the head in a frame with rubber bands to maintain proper orientation of the rat

to the sources of stimuli ~27~.

Exposure to Dichloromethane Matlinckrodt reagent grade DCM (99.9% purej was used. and two channels of a mass-flow controlled gas biending system were adjusted to deliver the desired concentrations One channel gated compressed air through a 4-1 container of DCM. the outflow of which was mixed with air from the other channel in a 500-ml flask. Outflow from the flask was routed through a 190-ml sampling bulb in the line just before entry into the rat exposure chamber. The sampling bulb provided a source of gas samples just before exit to the rat. The rat was restrained in a plastic restrainer with an enclosed front that served as z bead-only exposure chamber (27). The rats were dynamically exposed ~2500 ml/min total flowj to air or 5.000. 10.000. and 15.000 ppm DCM: at this flow rate ambient noise was not loud enough to mask other auditory stimuli. Gas concentrations were sampled from a needle in front of the rat's nose. and exposures were calibrated against standards by gas chromatography. The desired concentration was achieved in about 30 sec. Each rat was exposed to each concentration and served as its own control. Exposures for each rat were separated by about one week. After EP recordings were obtained during exposure to air only, the rats were exposed to DCM for 60 min. Before exposures, an electrophysiologic test battery (vida infraJ was run. and then a program for obtaining sequential somatosensory and flash EPs was begun, i.e.. a second somatosensory and flash EP were obtained just prior to the onset of the exposure. During exposure somatosensory and flash EPs were obtained at 5-rain intervals up to 45 rain. except when the general test battery was administered (after 25 and 50 min of exposure). The test battery was then administered at 5.30. and 60 min after the exposure was terminated.

Arterial Carboxyhemoglobin After the main experiment was completed, three rats received chronic femoral artery cannulas under pentobarbital anesthesia I65 mg/kg) and were tested several days later while exposed for 60 rain to 15.000 ppm DCM Approximately 0.4 ml of blood were obtained at various times during the exposures and analyzed on an IL Model 282 Co-oximeter.

Electrophysiological Tests A battery of electrophysiological tests (TSTBAT) and other individual test routines were used. TSTBAT Consisted of samples of the spontaneous EEG and sensory-evoked potentials elicited by clicks, light flashes, and brief electric shocks to the tail. The spontaneous EEG was recorded simultaneously from the somatosensory and visual cortices, and a single channel of EP data was collected for each stimulus type (somatosensory and auditory EPs from somatosensory cortex, visual and brainstem auditory EPs from visual cortex). Spontaneous EEG. Four consecutive 5-sec samples of EEG (500 data points) were obtained with a recording bandpass Of 1 to 40 Hz. Pip-evoked brainstem aud?tory-evoked response (PBAER). This response was elicited by 1.4-,nsec duration, 16~kttz tone pips (0.2 msec rise and fall), with alternating polarity, delivered through a tweeter (1.5 to 20 kHz) suspended 24 cm directly above :the rat's head (adding 0.8 msec to component latencies); Intensity w a s about 70 dB above the level at which PBAERs are just discernible in rats ( 26,31 ). PBAER averages (10-msec epoch) were based on

DICHLOROMETHANE, EEG, AND EVOKED POTENTIALS

1,000 pips presented at 18.8/sec, using a recording bandpass of 400 Hz to 6 kHz. The relatively high highpass setting improves the signal-to-noise ratio and the definition of early peaks. Cortical auditory-evoked potential (CAEP). Clicks of 100txsec duration were presented at a rate of 0.7/sec to elicit a CAEP. Intensity was about 85 dB above threshold. Averages were based on 75,400-msec epochs, using a recording bandpass of 1 to 100 Hz. A stimulus rate of 7.4/sec was used to elicit the steady-state response (1 to 40 Hz recording bandpass, 2-sec epoch). We examined this response to determine if, through neural entrainment and recruitment, a measure of acoustic-related cortical activity that was more reliable than the CAEP could be obtained. Flash-evoked potential (FEP). FEPs were elicited by a Grass PS-2 strobe lamp (intensity setting = 16) centered 20 cm above and 7 cm in front of the rat, angled toward the rat's face. FEP averages (500-msec epoch) were based on 50 stimuli presented at 0.37/sec, using a recording bandpass of 1 to 55 Hz. This low-pass filter setting reduces 60 Hz interference without distorting the waveform. Prior to the FEP test the chamber was dimly lit (14 FL). The light was extinguished 15 to 30 sec prior to obtaining the FEP and then turned on again following FEP and steady-state FEP acquisition (which took about 6 rain to complete). Obtaining the FEP during the earliest phases of dark adaptation seemed to enhance the P2N2P3 component complex. Flashes were presented at 7.4/sec to elicit the steady-state FEP (2-sec epoch). Somatosensorv-evoked potential (SEP). SEPs were elicited by 50-txsec duration, 3 mA, cathodal constant-current square waves applied at a rate of 1.3/sec via needles inserted into the mid-ventral aspect of the tail (cathode proximal). The recording bandpass was 5 to 250 Hz (200-msec epoch), and each average comprised 50 samples.

621

5 PBAER

P1

EPOCH (ms)

N2

N1

FEP

N3

P2 N3 N1

N4

Data Quantification and Analysis The four individual EEG samples obtained at each test were converted to frequency spectra (0 to 25 Hz) and the spectra were averaged. Integrated power in 4-Hz bands and total power were obtained from the averaged spectra. Evoked potentials were quantified by measuring peak latencies and peak-to-peak amplitudes. Steady-state EPs were also converted to frequency spectra (0.5 to 32 Hz) and power at prominent peaks was recorded. Repeated-measures analyses of variance (ANOVA) were carried out on the several measurements for each exposure level separately. The 3-level repeated-measures factor consisted of data from 1) the preexposure baseline, 2) the 50-min test during exposure, and 3) the test 60 rain after the end of exposure. Data from sequential acquisitions of the SEP and FEP are shown only for illustrative purposes. Because of the large number of EP variables analyzed and the theoretical potential for excessive type I errors, a 3-level repeated measures ANOVA, consisting of the three baseline sessions, was also run on EP data. This analysis provided an empirical estimate of the likelihood of obtaining significant differences by chance with the number of EP variables measured (41 variables). RESULTS

Exposure Levels and General Observations Average exposure levels after 45 min of exposure, just before the test battery was run, were 5,200, 10,100, and 15,100 ppm with average standard errors of 2.9%. The average exposure level for the hour, estimated from samples obtained at 5, 15, 25, 40, and 45 rain, were 5,800, 10,700. and 14,800 ppm. Colonic temperatures were 37.7, 37.8, and 37.3°C in the 5,000, 10,000, and 15,000 ppm conditions, respectively, at the 50-rain test. These were

P3

P4

FIG. I. Superimposed group-averaged EPs from the three baseline tests preceding each of the different concentrations of exposure.

- 0 . 3 , 0.0, and - 0 . 5 ° C different from their respective baseline temperatures--sufficiently alike to preclude any substantial effect on the EPs. After a period of weight loss following surgery, weights increased and were between 290 and 305 g for the remainder of the experiment. During exposures the rats were restrained and placed in a sound-attenuating chamber so direct observations during exposures were not possible. However, incidental observations during preliminary tests with rats removed early after the end of exposure indicated that these levels of exposure induced behavioral depression, ataxia, transient tremor, and sometimes gnawing of the extremities. In three cannulated rats exposed to 15,000 ppm DCM for 60 rain, average COHb at the end of exposure was 7.1%.

Baseline Comparisons of Evoked Potentials Group mean EPs for the three baseline conditions were virtually identical, as shown in Fig. 1. ANOVAs on FEPs (16 measures) and PBAERs (15 measures) revealed no significant (p<0.05) effects; mean F(2,22) ratios were 0.94 and 1.05 (ps = 0.41 and 0.37, respectively) for the FEP and PBAER, respec-

622

REBERT, MATTEUCCI A N D PRYOR

020

-

-

-

-

I)

(~% 0 5 K ppm (2 J- x 10 ' ) • • 10K pprn(1 3 ~ ,~ _) ii\ --~15K p p m ( l 3 :, !0 -'J,

015 co c 0

O @ O9

//1 /

i ~

o+os

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

+]+Sk F'P' •

.

•10;:~r.r:1

:

!

(2

4

.]-4+ × ~,a

f)

S x I 0 -~:)

O2O z

0 lf"

/

/

~ R " z,k q A

/

.

0 10

2~

0

O5

+

000

0 O0

Exposure ~?

Exposure

--17

0 O5

005

) 2C

O.46

035

l

030

I

8 o~+ I

~,~,

...............

+1

O---O 5K p p m ( 9 9 x !O ,-4) • --•1OK ppm ( 4 t x 10 - 6 ) /A - - A ! S K ppm(9.9 x 10 - 1 ] ) r

0,40 t P S oq ]o

.

'~s --Zs l ,K p ~ ] ( 4

,w ,:-s ~

[

.

P

2.....

010

5~

0 5t])

i

'E

O

P5

O5~4 •pro ~2 6 × 10 ~)

-

,

•

.

~ -O

co

+oo+i./S

---+-__+

0,00 j-

oosl -20

ff×Doqure

Exposure

v

. . . . .

0

20

v ~0

60

V

) OE 80

100

120

40

Minutes Relative to Start of Exposure

2r

V 2f

40

51~

Bit

Mpnutes Relatve tc ?tar!

~@U u

~ 2C

4(

if%••sure

FIG. 2. Latencies of PBAER components and P1 to P5 interwave time as a function of time relative to exposure and concentrauon of dichloromethane. In this and all subsequent figures, plotted values are mean differences t + SE from the baseline score, and the numbers opposite the plot symbols are p values from ANOVAs or t-tests.

tively; the highest Fs were 3.22 and 2.36. The latencies of the N2 and P2 components of the SEP (10 measures total) were 4 and 2% shorter, respectively, in the first test than during the last two tests and N2P2 amplitude was 14% larger; these differences were significant ( p s i 0 . 0 3 ) .

AuditoR-Evoked Potentials Brainstem auditory-evoked response. In the following we refer to as "significant," F-ratios with probabilities of occurrence less than 5%. Significance levels (shown in the figures) ranged from 4.83 × 10 - 2 to 9.97 × 10-~1. Seventy percent of the significant F-ratios for BAERs were associated with ps-<7.00 × 1 0 - 3 At all concentrations, DCM significantly increased the latencies of components 1, 3, and 5 and the 1-5 interwave time (Fig. 2). In each case the effect was concentration-related. The effect of 10,000 ppm was only slightly greater than that o f 5,000 ppm after 25 min of exposure; later in the exposure the effect of the 10,000-ppm concentration was greater than that of the 5,000-ppm exposure, but the extent of this difference depended on the particular component--being greatest for P3. Recovery toward normal values occurred, but it was not complete for the 15,000ppm exposure 60 min postexposure. The late exacerbation of effects on P5 latency and the P1-P5 time in the 10,000 ppm condition was not due to changes in body temperature; temperature was 37.8°C in the baseline run and 37.6°C at the 120 min test. BAER component peak-to-peak amplitudes, through peak N2P3, were significantly reduced by 10,000 and 15,000 ppm, and the effect was more pronounced for the highest concentration (Fig. 3). However, middle components (-N3P4-P4N4) increased in amplitude during exposure, although the changes in P4N4 were not concentration-related; the last components (N4P5 and P5N5)

were also reduced in amplitude by the t5.000 ppm concentration (Fig. 3). Cortical auditory-evoked potential. The cortical auditory EP. recorded from somatosensory cortex, consisted of a simple positive-negative sequence that was essentially abolished by all concentrations of DCM. Because this response was so completely disrupted, quantitative evaluation was not attempted. There was only slight recovery of this response 60 min after the end of exposure. There were few reliable changes in spectral parameters associated with repetitive acoustic stimulation at 7.4/sec: there was a tendency for spectral peaks near 15 and 22 Hz to be depressed by the 5.000 ppm exposure but enhanced by the higher concentrations ~Fig. 4~: other parameters were not changed by DCM.

Flash-Evoked Potential With exposure to 10.000 and 15.000 ppm DCM. there was an apparent decrease in the latencies of FEP components N1 and P2. However. as shown in Fig. 5. DCM gradually eliminated the N I component, so at the 50-rain test it was scored at the point of first deflection in the waveform, which occurred earlier than the real peak. The decrease in P2 latency appeared to be the result of eliminating influences related to the N1 component, perhaps uncovering the "'true" PI component. However. latency changes extended to some later peaks as well: P4 and N4 also significantly decreased in latency at the 5.000-ppm exposure (Fig. 6). At 15.000 ppm these peaks significantly increased in latency (Fig. 67 and then decreased below baseline values 5 min after the exposure was stopped. There was little change during the 10.000 ppm exposure, but response latency still declined below baseline when the exposure stopped. As shown in Fig 7. apparent latency effects on NI were evident within about 5 min of exposure onset at

DICHLOROMETHANE, EEG, A N D EVOKED POTENTIALS

623

0 40 +

P1N1

O--O5K

ppm (NS) 5 0--0 1OK ppm(61 x 10 7) ~--A15K ppm(1 2 x 1 0 - )

000 T

050 +

t f', 2F 3

0 O0

[

•

l

(n

5> 0 o

040 \

080

~ \

/

\~

~20 k

AExposure

, V~-

1 60

/

]

i

e/J

T

~ /

150

I

.L

:

,

20q

O

T

O0

7

O 5K ppm

•

[

/ O

•

~oK

(NS) ( '

O--05Kppm •--• Tg--A

P4N4

J\

/:i<

?,

\Tj!J

,

(NS )

1OK pprn(4 8 x 10 - 2 ) 5Kpp~(~7,~o-~j

/r

+

0 O0

O--OSK ppm (! 7 × 10 - 2 ) • --• 10K pprr, (,NS) 3 A Z~!SK ppm(28 × } 0 - )

7

0 50

+

:4iPi<;

000

,

050

V

Exposure ,

,

V

,

,

t

[ .

I V

Exposure

V

*'

0.50

075

050 N4P5

P5N5

0 5O 0 25

T

000

%---.__ TJ

t

•

±

025 0.50

0 50

O75

075 i O•

I 25 - - , -20

t

025 +

00 025

i

Exposure . . . . .

1.00

0 5O

.00

N+-4

150 N,3P4

0 > 0L <)

/

1 O•

50

0 > 0 L_

\\T

0.50

[xposure

7 0

.z-, 20

O--• 10K ppm (NS) Z~.--- S 15K ppm(5 x l 0

_m J)

V 40

6(]

1 O0 125

80

100

120

Minutes Re otive to Start of Exposure

140

20

0 Minutes

20

40 Relative

60

80,

to Stori

of

: O0

12o

E~,;L,

urr,

'40

FIG. 3. Amplitudes of PBAER components as a function of time relative to exposure and concentration of dichloromethane.

15,000 ppm, whereas N4 began to change only after 15 to 20 rain. The latter time-course was the same for the P4 component. In comparing N4 latency in Fig. 6 and 7 the magnitude of the latency effect is greater in the latter. This is due to two factors: 1) perhaps because the baseline test for the sequential EPs was executed very shortly after the somatosensory test during TSTBAT, latencies of P4 and N4 components of the flash EP were shorter than in the first baseline test; 2) the latency effect appeared to be maximal at 40 minutes, decreasing somewhat by 45 minutes and perhaps more so when TSTBAT was run at 50 rain. Components PI and N1 were abolished by DCM (Fig. 5). As shown in Fig. 8, NI amplitude was affected about equally by the two highest concentrations, essentially disappearing, but with full recovery. The effect was less at 5,000 ppm, with overshoot above the baseline value during the recovery phase. Amplitude measurement of the N1P2 and P2N2 waves was meaningless because of the loss of NI and the "uncovering" of the "true" P1 wave. The N2P3 wave significantly decreased in amplitude at the two highest concentrations (not significant at 5,000 ppm), and it exhibited little recovery (Fig. 8). Of the later waves (P3N3-P4N4), the only

systematic effect during exposure was a significant decline in P4N4 amplitude associated with the exposure to 15,000 ppm DCM (Figs. 5, 8). Peaks in the spectra of the repetitively driven FEP tended to be reduced by DCM but the effect was not systematically related to exposure concentration (data not shown).

Somatosensoo'-Evoked Potential As seen in Fig. 9 the NI component of the SEP exhibited multiple wavelets (e.g., N l a and N l b ) suggesting that it comprised two or more independent components. Preferential depression of N l b amplitude gave the impression in most individual cases that the latency of N1 decreased, but that was not substantiated by further evaluation; the latency of N l a in the groupaveraged SEP was constant throughout the exposure period and was the same at all concentrations of exposure. Similarly, as the N l b and P1 components shrank, the P1 peak appeared to decrease in latency as well. N2 latency significantly decreased during exposure to 5,000 ppm, but was not significantly altered during exposure to the higher concentrations (Fig. 10). There were no

624

REBERT, MATTEUCCI AND PRYOR

consistent or concentration-related changes in the latencies of components P2 and N3. The amplitudes of all SEP components were significantly depressed by all concentrations of DCM ( p s - 1 . 5 × 10 ~- to 2.1 × 1 0 - ~o), and this effect was evident at the 5-min exposure test, e.g., changes in the amplitude of component P2N3 at 5-min intervals during exposure are shown in Fig. 11. An example (component N2P2) of amplitude changes for the various concentrations and phases of the experiment is also shown in Fig. 11. There was a concentration-related decrease in amplitude with full recovery after 60 min postexposure for exposures of 5.000 and 10,000 ppm; recovery was not complete for the 15.000 ppm exposure, Changes in the SEP were not the result of alterations of peripheral nerve. Compound action potentials recorded from the ventral caudal (tail) nerve in 6 rats remained almost normal throughout a 20-min exposure to 15,000 ppm D C M - - a m p l i t u d e decreased by 14% and latency by 11%.

7"~

J:~,,_ •

L,L, r"

~,j

"t

•

•

" ("

: t~,~=

t'.-,

Electroencephalogram In both somatosensory and visual cortices the main effect of DCM on the EEG was to increase power in the 8-12 Hz and 12-16 Hz frequency bands (Fig. 12). This effect was concentration- and time-related. It was apparent at the 25-min test for 10.000 and 15,000 ppm, but not as evident until the 50-rain test with 5.000 ppm. However, except for the 8-12 Hz band, 25-rain test. at 5.000 ppm, all means during exposure differed significantly from the preexposure values. There was only moderate recovery from this effect in the time span examined for the 8- to 12-Hz band. whereas recovery did occur in the 12- to 16-Hz b a n d - - i n fact. in the latter. a decrease from peak power occurred before the 15.000 ppm exposures were over.

FIG. 4. Spectral power at 14.6 ~upperJ and 22 Hz in the EEG "'driven" by repetitive clicks as a function of time relative to exposure and concentration of dichloromethane.

lO,O00ppm

15,000 ppm •

"" Relative to of Exposur 45

40

20 15 10

-10 (Baseline) P3

P4 After-Discharge

FIG. 5. Group-averaged FEPs obtained several times during exposure to 10,000 and 15,000 ppm dichloromethane~ At both concentrations the N1 component was abolished, but only at 15,000 ppm was there a substantial effect on the P4N4 wave.

DICHLOROMETHANE, EEG, A N D EVOKED POTENTIALS

625

DISCUSSION

The evaluation of a large number of variables in an experiment is often a source of concern because of the theoretical possibility of increasing the likelihood of type I statistical errors (17). However, in several experiments in which multiple EP baseline sessions have been run, EP group mean waveforms have been virtually identical [e.g., (27)]. That was also true in this investigation. One approach to controlling the overall experimentwise probability of type I errors is to carry out a comprehensive M A N O V A before undertaking specific comparisons. Instead, we examined the empirical outcome of analyzing 41 variables (not all of which were independent) across the three baseline tests. We found 7.3% (3/41) of the analyses to be significant using an alpha criterion of 5%. Thus, this empirical estimate of type I errors suggests that the results of exposures were not due to an excess of spuriously significant comparisons. This is also supported by the very high levels of significance achieved in many of the ANOVAs. We estimated from three rats that a COHb level near 7% was produced by the one-hour exposure to 15,000 ppm DCM. As noted by Ciuchta et al. (6) COHb levels continue to rise postexposure--they reported a COHb level of about 9% 2 hr after the end of a 1-hr exposure to 5,000 ppm DCM. However, as shown by McKenna et al. (13), the conversion of DCM to carbon monoxide is a saturable metabolic pathway. Those authors did not observe a dose-related increase in blood COHb beyond 500 ppm; the level observed at DCM concentrations of 500 or 1,500 ppm after 1 hour of exposure was about 7.5%. Therefore, it is unlikely that any electrophysiological effects that we observed were due to carbon monoxide. It has been reported that rather severe levels of hypoxia are necessary to produce changes in EPs (32,34), and we found little, if any, effect on several EPs of rats when, during recovery from exposure to 0.2% CO ( C O H b - 7 0 % ) , arterial COHb was about 30%; even during the severe exposure the EPs were only

~°I

I

T

2~

i I

39

'-)O 5K pprT (1 4 × 1'3 4 ) • - - - • 1 0 K p p m ( 7 3 × IC I ," - - " /~ [,K p p r n l 5 / ~ ~C JI

c oF

lit

1D

" -~

1

'k ,,~\T.

Ex_,posu~e

• CO

I

U] I

T~ C

sq

:

~5Kppm

~C [

•

• 1OK ppm(1 1 ~ 10 ~}

: 6} .

~'~

z~

A 15K ppml',3 #]i x

(44~

!0

~:

s !

,I )

"

£.

£

1

2:3

,L

Expc, s s~e - 20

3

"¢! r,,.te:

::0

4©

60

80

100

12,0

!40

A
FIG. 6. Latency of FEP late components P4 (upper) and N4 as a function of time relative to exposure and concentration of dichloromethane.

o 69

) bK ppm •1OK ppm ,t~ 15K p p m

O

5~

÷h c,

• h

T t

si 10 "5 1

2O

v

1

25

14r,

%4

0

12,3,

::,Sk ;pro • 10K Spin, ,', i 5 k rsprr"

• zX

1 O0 cc E o L~ © o• •

80 [ ~-

60 :

,

A

4 ',)

A

s,?l +

19

0 U:nu!es

1 (} C?e C t ] v ' e

•

20 to

~", ~{" ~ ' t

c'

4q

5N

[xs;osbre

FIG. 7. Latency of FEP components NI (upper) and N4 as a function of time during the exposure period and concentration of dichloromethane. Changes in N1 were evident within 5 min during exposure to 15,000 ppm, but P4 latency was not clearly altered until after 15 to 20 rain.

moderately affected (25). These results are similar to those reported by Petajan et al. (19). Also, Dyer (9) observed no significant changes in EPs recorded from the rat's superior colliculus until COHb reached 22%. Dichloromethane did not affect the several sensory systems in the same way. It increased almost all BAER component latencies and interwave times at all concentrations, but whether it increased or decreased FEP late component latencies depended on concentration (the specific effect on FEP P1N1 amplitude precluded a meaningful evaluation of FEP early component latency changes). Because of changes in the morphology of the early part of SEPs it was also difficult to reliably interpret any changes in those peaks as due to shifts in latency. However, an intermediate (N2) peak was altered, whereas later peaks were not; this contrasted with the FEP--its latest peaks (P4,N4) were affected (but, vida infra). Amplitude changes in the FEP and BAER depended on the specific components considered. Whereas early and late components of the BAER decreased in amplitude, the middle components were enhanced. Although DCM did not affect the amplitudes of all FEP components, its effect was always to decrease amplit u d e - n o components were enhanced. DCM affected the SEP in a more consistent w a y - - a l l SEP components decreased in amplitude. In this respect the change in the CAEP was like the SEP. Because of the relatively small changes sometimes observed at the 5,000 ppm concentration (e.g., FEP N4 latency, Fig. 8), it is possible that they might reflect effects of temporally repeated testing and that it would be useful to examine the rats sequentially throughout a zero-ppm sham exposure. We did not include such a condition in this experiment, partly on the basis of the 25% additional effort entailed, but primarily because we had developed considerable confidence in the stability of most of the EPs over the course of repeated tests within a daily session. Our comparison of the several baseline tests in this experiment gave some support to

626

REBERT. MATTEUCCI AND PRYOR 120

MinRelativeto

N1

Start of Exposure

O--05K 0--010K A--&15K

8O

ppm ( 3 2 x 10 - 6 ) ppm(4.0 x 10 - 9 ) ppm(2.6 x 10-~1

,

~

0

4O

o > 0 £)

+

40

o

4O

~/~k

. / ~ / . / ~

20

8O 120 ___.

V

.

.

Exposure .

.

.

V

.

.

.

.

.

.

.

.

.

2O 10

l

o

•

N2P~

O--05K ppm (NS) 0 - - - 0 1 O K pprn(2.6 x 1 0 - 2 ) A--A 15K ppm(5.5 x 10 - 3 )

+ u~ go +,

10

0> o o

20 50 40 5O

(Baseline) 1

60 7O _ ~

V

,

Exposure ,

,

,

.

P2

~Z

120

P4N4

d)

40

o >

+

I

o

FIG. 9. Group-averaged SEPs obtained at 5- to 10-min intervals during exposure to 10.000 ppm dichloromethane The solvent caused all components to decrease in amplitude. Changes in the latencies of early components were difficult to assess becau~ of changes in the morphology of the waveform associated with subcomponents N1 a and Nlb.

O--05k ppm (NS) 0--0 IOK pprn (NS) } ~ - - Z ~ 15K pprn(1.4 x 10-" )

80

I

T

I

[

0

(D

i %.

&_J~/

o

T

~x~

&,,,

40 80

/ &

Exposure 120 2O

0 Minutes

20

40 Relative

60 to Start

80

100 of

120

i40

Exposure

FIG. 8. Amplitudes of FEP components as a function of time relative to exposure and concentration of dichloromethane.

that confidence. Additionally, Rebert et al. (27) showed that several group-averaged EPs obtained from sham exposure sessions were essentially identical to one another. Preliminary data (8 rats) from a study in progress, in which a sham exposure condition is included, showed almost the same result. For group-averaged SEPs the zero lag waveform cross-correlation with an initial group-averaged SEP, of waveforms obtained at 25, 55, 65, 90, and 120 min after the initial acquisition, ranged from .93 to .97. For PBAERs the correlations ranged from .96 to .981 FEPs were less stable. We have observed a tendency for late component latencies to decrease somewhat over the course of testing. For the group-averaged FEPs, correlations ranged from .64 to .92. For the most part, then, the significant changes associated with 5,000-ppm exposures can be considered valid, but another consideration is also important. In many instances where the 5,000ppm condition caused a significant deviation from the preexposure

value there was a return toward normality in the recovery plmse: that is not a pattern that would be expected from repeated testing per se. However. when the pattern does not occur, the interpretation is made more difficult. For example, it is very possible that the significant decreases in FEP P4 and N4 latencies (Fig. 6) were not related to the exposures. Because of the tendencies for "'spontaneous" change we have observed in the FEP. we conclude that a sham-exposure condition would improve the interpretation of changes in that response. One of the most interesting findings of this study was the fairly specific obliteration cat 10.000 ppml of the FEP N l component. It appears from this result that the components typically labeled " P l " and " P 2 " ' are not independent entities, but are the initial descending and subsequent ascending limbs of the " t r u e " P1 component, interrupted by NI The N1 component is generally considered to reflect the initial excitatory barrage of corticofugal tmpulses from the lateral geniculate nucleus (4t. One might suspect that subsequent components reflect additional intracortical activities consequent to the initial input. However. at 10.000 ppm DCM. there were no significant decreases m the amplitudes of component P3 or later, although NI was gone. This is consistent with the suggestion that the later waves reflect the influences of subcortical regions other than the lateral geniculate (4). There is some question about the relationship of the N3 wave to subsequent after-discharges N4 and later waves (4k the results at 15,000 ppm suggest independent generators because N3 remained normal. whereas the after-discharge disappeared. The inter- and intramodality specificities observed here sugges~ that DCM affects some specific neurochemical mechanism that varies in its influence among neural systems. Such a notion is

DICHLOROMETHANE,

EEG, AND EVOKED

POTENTIALS

627

o

C 7'

l

A

,/ hi'

• /

' ',)~ ; [:'T71 4 ~ ] C ' + :: ~ll , I

.L

t

I

;(

O~

• L

•

t

i

]

& ! 'l" !;u'-

,HK

" )0

i{

11

i

1, , K ' i

I 4 ;,9

FIG. I 0 Latency of SEP componem N2 as a function of time relative to exposure anu concenlratmn of dichloromethane.

t

i'

reinforced by the differences in the effects of DCM and toluene. For example, while DCM had essentially the same effects as toluene on BAER component latencies, the pattern of changes in amplitudes among components were just the opposite. Also. whereas toluene, at some exposure parameters, greatly enhanced S E P component amplitudes ,and differentially so~. DCM uniformly caused SEP components to decrease in size. Toluene had no effect on spectral peaks induced by repetitive acoustic stimulation, but DCM tended to enhance those peaks. With respect to the FEP, toluene increased the amplitudes of early components. eliminated late components, induced oscillations in visual cortex. and had no discernible effects on component latencies. In contrast.

f

i ::

~i.{

] :

• '

/

^,

"

--

Sk

aEn,

,/~a 1 .%1~

7;F, rr

i

.... ;. 4 : {

I

! 12', i

-

.

.

.

.

.

i~

.

.

.

.

.

:

; )

. 51;

.1{I

: ; sk ~*prl :4 o ~ ' i "~f, • 1CK r'p,'l~8 ~ "0 ; :) *~k ppm(~-; . 11} '; t

• ' F

L[

J i

• l

(} ,!

i

• ;c; !

]2:

,

'.!r

•,

~iv

,i.

,1:

-~i:'i.,

,

":,

11:(2

L~,,

:tcrt

,,f

1 i}

"4(

F~r_;::;.cl•re

FIG. I 1. Amplitudes of SEP components P2N3 (upper) and N2P2 as a

function of time relative to exposure and concentration of dichloromethane. The amplitude of component P2N3 is shown for several intervals during exposure and the amplitude of component N2P2 is shown for the different phases of the experiment.

2"

:,~i J

i 'C'

L 4

['~r

]~

: ",i

::

~, :,

i ,],!

]

,

" i .[,

FIG. 12. Spontaneous EEG integrated spectral power in 8 to 12 Hz (upper) and 12 to 16 Hz frequency bands as a function of time relative to exposure and concentration of dichloromethane.

at 10,000 ppm, DCM eliminated the N1 component, had little or no effects on amplitudes of the later components, did not induce oscillations, and possibly affected some component latencies. Thus, in many respects the effects of these two solvents appear antagonistic. A physiological antagonism of toluene and DCM has been noted before with respect to the metabolism of DCM to carbon monoxide: almost no COHb was evident in DCM-exposed rats pretreated with toluene (6). Behaviorally, toluene and DCM have different profiles of effects and appear to act additively in that regard (22). Also, whereas toluene prolonged postrotational nystagmus in rats, DCM shortened it (36). Because solvent mixtures are more commonly used than pure solvents, and toluene and DCM are common components of solvent mixtures, it will be important to determine how these two solvents interact electrophysiologically. It is clear that some specific predictions could be made from our independent studies of these chemicals, e.g., at the right combination it is possible that BAER and SEP component amplitudes could be unaffected, whereas changes in the latencies of BAER components would be exacerbated by the combination. Because toluene reduces the size of FEP late components and DCM eliminates P1NI, the FEP might be abolished by the combined solvents. Although industrial solvents, like inhalation anesthetics, at high concentrations cause sedation and anesthesia, the results of our studies and others [e.g., (10)] indicate that various solvents do not necessarily induce their general effects through the same mechanisms. This point was also emphasized, with respect to anesthetics, by Winter (38). There is a large literature concerning the pharmacologic effects of drugs on EPs, but without undertaking specific comparisons of various drugs and solvents in our test battery, it is difficult to draw conclusions about the neurochemical bases of the effects we have observed• Dyer et al. (10/ reviewed the effects of several drugs on the rat's FEP and noted that none mimicked toluene and p-xylene. Given the considerable specificity

628

REBERT, MATTEUCCt AND PRYOR

in the effects o f particular solvents both within (e.g., on specific E P components) and between sensory modalities, c o m m o n mediation through " n o n s p e c i f i c " reticulothalamic m e c h a n i s m s is unlikely. This is also clear from changes in amino acids in different parts o f the rat's brain consequent to D C M exposures, e.g., glutamate and g a m m a amino butyric acid decreased in frontal cortex but increased in cerebellar vermis (5). The situation is complicated by the fact that some effects may be mediated by the solvent itself, and others via a metabolite, e.g., Mattsson et al. (15) suggested that " e x c i t a t o r y " properties o f toluene were mediated by the metabolite o-cresol.

EP analysis has proven useful in characterizing differential C N S effects o f several solvents, and at the same time, some solvent effects have been informative with respect to the nature o f the c o m p o n e n t structure o f the EPs, e . g . , the independence o f FEP N1 from other components, and the existence o f a " t r u e " FEP PI component. Whereas elucidating the acute effects o f solvents on the nervous system is important to a full understanding o f their abuse potential and for reasons o f safety, we have not found that long-term effects necessarily mimic acute responses. For example, long-term exposure 1o toluene had little effect on a similar battery o f EPs (21), whereas the acute effects are quite profound (28,29/.

ACKNOWLEDGEMENTS The authors thank Dr. Charles Sharp of the National Institute on Drug Abuse (NIDA) for his encouragement and support of this work, and Rosie McCormick for preparation of the manuscript. This work was supported by NIDA Contract 271-87-3132.

REFERENCES 1. Alexeeff, G. V.; Kilgore, W. W. Learning impairment in mice following acute exposure to dichloromethane and carbon tetrachloride. J. Toxicol. Environ. Health 11:569-581; 1983. 2. Annau, Z. Neurobehavioral toxicology. Baltimore: The Johns Hopkins University Press; 1986. 3. Barrowcliff, D. F.; Knell, A. J. Cerebral damage due to endogenous chronic carbon monoxide poisoning caused by exposure to methylene chloride. J. Soc. Occup. Med. 29:12-14; 1979. 4. Bigler, E. D.; Neurophysiotogy, neuropharmacology, and behavioral relationships of visual system evoked after-discharges: A review. Biobehav. Rev. 1:95-112; 1977. 5. Briving, C.; Hamberger, A.; Kjellstrand, P.; Rosengren, L.; Karlsson, J. E.; Haglid, K. G. Chronic effects of dichloromethane on amino acids, glutathione and phosphoethanolamine in gerbil brain. Scand. J. Work Envimm Health 12:216-220; 1986. 6. Ciuchta, H. P.; Savell, G. M.; Spiker, R. C., Jr. The effect of alcohols and toluene upon methylene chloride-induced carboxyhemoglobin in the rat and monkey. Toxicol. Appl. Pharmacol. 49:347-354; 1979. 7. Dick, R. B. Short duration exposure to organic solvents: the relationship between neurobehavioral test results and other indicators. Neurotoxicol. Teratol. 10:39-50; 1988. 8. Dyer, R. S. The use of sensory evoked potentials in neurotoxicology. Fundam. Appl. Toxicol. 5:24--40; 1985. 9. Dyer, R. S. Effects of prenatal and postnatal exposure to carbon monoxide on visually evoked responses in rats. In: Merrigan, W. H.; Weiss, B., eds. Neurotoxicology of the visual system. New York: Raven Press; 1980:17-33. 10. Dyer, R. S.; Bercegeay, M. S.; Mayo, L. M. Acute exposures to p-xylene and toluene alter visual information processing. Neurotoxicol. Teratol. 10:147-153; 1988. 11. Dyer, R. S.; Jensen, K. F.; Boyes, W. K. Focal lesions of visual cortex--effects on visual evoked potentials in rats. Exp. Neurol. 95:100-1t5; 1987. 12. Leukroth, R. W., Jr. Predicting neurotoxicity and behavioral dysfunction from preclinical toxicologic data. Bethesda, MD: FASEB; 1986. 13. McKenna, M. J.; Zempel, J. A.; Braun, W. H. The pharmacokinetics of inhaled methylene chloride in rats. Toxicol. Appl. Pharmacot. 65:1-10; 1982. 14. Mattsson, J. L.; Albee, R. R. Sensory evoked potentials in neurotoxicology. Neurotoxicol. Teratot. 10:435--443; 1988. 15. Mattsson, J. L.; Albee, R. R.; Gorzinski, S. J. Similarities of toluene and o-cresol neuroexcitatiou in rats. Neurotoxicol. Teratol. 11:71-75; 1989. 16. Mitchell, C. L. Nervous system toxicology. New York: Raven Press; 1982. 17. Mueller, K. E.; Otto, D. A.; Benignus, V. A. Design and analysis issues and strategies in psychophysiological research. Psychophysiology 20:212-218; 1983. 18. Pankow, D. R.; Gutewort, R.; Glatzel, W.; Tietze, K. Effect of

19.

20.

2l.

22. 23.

24. 25.

26. 27.

28.

29. 30.

31.

32.

33.

dichloromethane on the sciatic motor conduction velocity of rats. Experientia 35:373-374: 1979. Petajan, J. H.: Packham. S. C.: Frenas. D. B.: Dinger, B G. Sequelae of carbon monoxide induced hypoxia in the rat. Arch. Neurol. 33:152-157: 1976. Pryor. G. T.: Dickinson. J.: Feeney, E.: Rebert. C. S. Hearing loss in rats first exposed to toluene as weanlings or as young adults. Neurobehav. Toxicol. Teratol 6:111- l 19: 1984. Pryor. G. T.: Dickinson. J.: Howd. R. A.: Rebert. C. S. Neurobehavioral effects of subchronic exposure of weanling rats to toluene or hexane. Neurobehav. Toxicol. Teratol. 5:47-52: 1983. Pryor. G T.; Rebert. C. S. Neurotoxicity of inhaled substances. Quarterly Report No. 5. N1DA Contract 271-87-3132. Menlo Park. CA: SRI International; 1988. Pryor, G. T. Pharmacological neurohehavioral aspects or solvent toxicity based on animal studies. WHO Advisory Group Meeting on Adverse Health Consequences of Volatile Solvents/Inhalants, Mexico City, Mexico, April. 1986. Rebert. C. S. Multisensory evoked potentials in experimental and applied toxicology. Neurobehav. Toxicol. Teratol. 5:569-671: 1983. Rebert. C. S. Neurobehavioral incapacitation of rats following exposure to carbon monoxide and carbon dioxide. Final Report, NBS Grant 6ONANB6DO644. Menlo Park. CA: SRI International: 1987. Rebert. C. S.: Becker. E. Effects of inhaled carbon disulfide on sensory-evoked potentials of Long-Evans rats. Neurobehav. Toxicol. Teratol. 8:533-541: 1986. Rebert. C. S.; Davis. E. E.: Juhos. L. T.: Jensen. R. A.: Pry.or. G. T.: Robin. E. D. Effect of acute respiratory acidosis on multimodality sensory evoked potentials of Long Evans rats. Int. J. Psychophysiol. 8:155-168: 1989. Rebert. C. S.; Matteucci. M. J.: Pryor. G. T. Multimodal effects of acute exposure to toluene evidenced by sensory-evoked potentials from Fischer-344 rats. Pharmacol~ Biochem. Behav. 32:757-768: 1989. Rebert. C. S.: Matteucci. M. J.: Pryor, G. T. Acute electrophysiologic effects of inhaled toluene on adult male Long-Evans rats. Pharmacol. Biochem. Behav. 33:157-165: 1989. Rebert, C. S.: Schaeffer. J. A.: Dahlgren. J. G. Sources of exposure to neurotoxicants: neuropsychological and electrophysiological consequences. In: Ellingson. R J.; Murray. N. M. F.: Halliday, A. M.. eds. The London Symposia. Amsterdam: Elsevier; 1987:355-359. Rebert. C. S.: Sorenson. S. S.: Howd, R. A.: Pryor. G. T. Toluene-induced hearing loss in rats evidenced by the brainstem auditory-evoked response. Neurobehav. Toxicol. Teratol. 5:59-62: 1983. Rebert. C. S. Changes m sensory evoked potentials associated with compromise of cerebral metabolism. Navy Workshop on the Effects of Combined Fire Products on Human Physiological and Psychological Performance. Groton. CT. November. 1987. Sharp, C. W.: Brehm. M. L. Review of inhalants: Euphoria to

D I C H L O R O M E T H A N E , EEG, A N D E V O K E D POTENTIALS

dysfunction. Washington, DC: U.S. Government Printing Office; 1977. 34. Sohmer, H.; Freeman, S.; Malachi, S. Multimodality evoked potentials in hypoxaemia. Electroencephalogr. Clin. Neurophysiol. 64: 328-333; 1986. 35. Stewart, R. D.; Fisher, T. N.; Hosko, M. J.; Peterson, J. E.; Baretta, E. D.; Dodd, H. C. Experimental human exposure to methylene chloride. Arch. Environ. Health 25:342-348; 1972.

629

36. Tham, R.; Bunnfors, I.; Eriksson, B.; Larsby, B.; Lindgren, S.; Odkvist, L. M. Vestibulo-ocular disturbances in rats exposed to organic solvents. Acta Pharmacol. Toxicol. 54:58-63; 1984. 37. Winneke, G. The neurotoxicity of dichloromethane. Neurobehav. Toxicol. Teratol. 3:391-395; 1981. 38. Winter, W. D. Effects of drugs on the electrical activity of the brain: anesthetics. Annu. Rev. Pharmacol. Toxicol. t6:413-426; 1976.