JOURNAL OF APPLIED TOXICOLOGY, VOL. 12(5). 335-342 (1992)
Diethylbenzene Inhalation-induced Electrophysiological Deficits in Peripheral Nerves and Changes in Brainstem Auditory Evoked Potentials in Rats F. Gagnaire,? M. N. Becker, B. Marignac, P. Bonnet and J. De Ceaurriz Institut National de Recherche et de Securite. 54501 Vandoeuvre, France
Key words: diethylbenzene; neurotoxicity; brainstem auditory evoked potentials; nerve conduction velocity; rats; inhalation exposure.
Motor and sensory conduction velocities (MCV and SCV), amplitude of the sensory action potential (ASAP) of the tail nerve and parameters of brainstem auditory evoked potentials (BAEP) were studied in male Sprague-Dawley rats after prolonged inhalation exposure to a commercial isomer mixture of diethylbenzene (DEB mixture) containing 6% 1,2-DEB. The MCV, SCV and ASAP were studied in one control group (10 rats) and three groups of 12 rats exposed to 500, 700 or 900 ppm DEB mixture for 6 h daily, 5 days per week, for 18 weeks. Rats used for recording BAEP (one control group and two other groups of 15 rats) were exposed to 600 and 800 ppm DEB mixture. The exposure time was the same. Rats exposed to DEB mixture exhibited a time- and concentration-dependentdecrease in MCV, SCV and ASAP and a time- and concentration-dependentincrease of both the peak latencies of all BAEP components and the interpeak (I-V) differences.
INTRODUCTION
MATERIALS AND METHODS
It was recently shown' that the commercial isomer mixture of diethylbenzene (DEB mixture), an intermediate of divinylbenzene synthesis,' administered to rats by oral gavage (750 or 500 mg kgg' once daily, 5 days per week, for 10 weeks) produced a timedependent decrease in motor and sensory conduction velocities (MCV and SCV) and in the amplitude of the sensory action potential (ASAP) of tail nerve. Rats treated with DEB mixture exhibited a ' blue discoloration of tissues and urine and presented evidence of clinical neuropathy leading to weakness of hind limbs and then paralysis. Oral administration of each separate isomer indicated that 1.2-DEB was the isomer responsible for neurotoxicity. Moreover, oral administration of 1.2-DEB produced a time-dependent increase of both the peak latencies of all brainstem auditory evoked potentials components (BAEP) and interpeak (I-V) differences and a decrease in the amplitude of the waveform.3 To our knowledge, the effects of long-term exposure have not been studied. The purpose of this study was to determine whether the same DEB mixture could cause peripheral and central sensorimotor neuropathies in rats when it was administered by inhalation at the three concentrations of 500, 700 and 900 ppm for a prolonged period of time. The integrity of the peripheral nervous system was investigated by measuring MCV, SCV and ASAP on the tail nerve and the integrity of the central nervous system was studied by using the brainstem auditory evoked potential (BAEP) technique.
Materials
t Author t o whom correspondence should be addressed. 026~437Xi921050335-OX$09.00 Wiley &L Sons. Ltd.
0 1991 by John
Diethylbenzene mixture (containing ca. 6% of 1,2DEB, 66% of 1,3-DEB and 28% of 1,4-DEB) was supplied by Aldrich Chemie, Steinheim, Germany. Animals
Ninety-one adult male Sprague-Dawley rats (IFFA Credo, Domaine des Oncins, Saint-Germain-sur1'Arbresle) were divided into two experimental groups A and B. Food (UAR-Alimentation, Villemoisson) and tapwater were available ad libitum. The rats were maintained under controlled environmental conditions with a 12-h dark/light cycle. Experimental design
In experiment A, 46 9-week-old rats were assigned to three exposed (12 rats each) and one control (10 rats) groups. The experimental groups were exposed to 500, 700 and 900 ppm DEB mixture for 6 h daily, 5 days per week, for 18 weeks. The control group was exposed to clean filtered air. The MCV, SCV and ASAP were measured every 2 weeks. At the end of the exposure period, the animals were kept for observation and neurophysiological measurements during a 6-week recovery period. In experiment B, 45 19-week-old rats were assigned to two exposed and one control group of 15 rats each. The two experimental groups were exposed to 600 or 800 ppm DEB mixture for 6 h daily, 5 days per week, Receiid 11 Ocrober I991 Acrepled (reimised) I N Fehruury 1992
336
F. GAGNAIRE ET A L . B
P
"1
700
650
600
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500
450 0 1 2 3 4 5 6 7 8 9 10 11 11 13 14 15 16 17 I8 19 20 21 22 23 24
0 1 2 3 4 5 8 7 8 910111213141S1017181920212223242S
Figure 1. Effect of DEB on body weight. (A) Rats were exposed to 500 (O), 700 (0) or 900 (A)pprn of a mixture of three isomers of DEB for 6 h daily, 5 days per week. (B) Rats were exposed to 600 (0) or 800 (A)pprn of a mixture of three isomers of DEB for 6 h daily, 5 days per week. The control groups were exposed to clean filtered air (0).In parentheses: number of ratr alive (see text). The arrows indicate the beginning of the recovery period. Week 0 values are before exposure.
for 18 weeks. The control group was concurrently exposed to clean filtered air. The BAEP were recorded every 2 weeks during the first 9 weeks of exposure and every 3 weeks after. The survivors were kept for observation and neurophysiological measurements during a 7-week recovery period. Generation of test atmospheres
Exposures were conducted in 200-1 stainless-steel inhalation chambers with adjustable airflow (1C30 m3 '-I)' To prevent leakage, were maintained at negative pressure (2-3 mmH,O). An additional airflow was through the DEB mixture and Output vapour was with air to the required concentration before entering the exposure chamber. Atmosphere sampling and analysis
The levels of exposure to DEB were controlled by periodically pumping a measured volume of the test atmosphere through a glass tube packed with active charcoal. The DEB was desorbed from the activated charcoal with carbon disulphide. The resulting samples were then analysed for DEB using a 1 mx118 in. column filled wiih chromosorb W impregnated to 15% with 20 M polyethyleneglycol and heated to 90°C. The proportion of each isomer in the atmosphere samples was checked every 2 weeks for each concentration. The analytical procedure required the use of a 6 m x U 8 in. column filled with chromosorb W
impregnated to 20% with tris-cyanoethoxypropane and heated to 100°C. The proportions of the three isomers were 5.9 ? 0.4, 66.1 2 1.1 and 27.9 2 0.9% for 1,2-, 1,3- and 1,4-DEB, respectively. Surgical procedures
Implantation of electrodes was carried out under sodium pentobarbital anaesthesia (30 mg kg-', i.p.); xylazine (4 mg kg-, , i.p.) was used to tranquilize the animals and reduce the dose of pentobarbital. Electrodes consisted of three stainless-steel Screws (1.2 mmx2.5 mm); The reference electrode was placed 3 mm posterior to the lambda. The ground electrode was;placed over the nasal bone 2 mm anterior to the nasal suture and mm to the midline on the right. The active electrode was placed 2 mm posterior to bregma and 6 mm to the right of the midline. The screws were connected to a microconnector (Connectral) and the apparatus was cemented to the skull with dental acrylic. Each rat was given 50 mg of ampicillin (Totapen) and allowed to recover from the effects of surgery for at least 7 days before starting electrophysiological recordings. Neurophysiological measurements
The animals were kept at least 16 h in fresh air before the electrophysiological measurements, except after weekends when 48 h elapsed between the end of
337
INHALATION EXPOSURE T O DIETHYLBENZENE
40
A
T
35
30 Y
>
Y
25 I
0 1
3
5
7 10 12 Duration of exposure (weeks)
14
16
18
1
20 22 24 Recovery period (weeks)
B T
25 0 1
3
5
7 10 12 Duration of exposure (weeks)
14
16
18 20 22 24 Recovery period (weeks) C
250
T
T
0 1
3
5
7
10 12 Duration of exposure (weeks)
14
16
1
18 20 22 24 Recovery period (weeks)
500 ppm, (0) 700 ppm, (A) Figure 2. Effects of DEB on the MCV, SCV and ASAP. (0)control group; DEB-exposed groups: (0) 900 ppm. Week 0 values are before treatment. The arrow indicates the beginning of the recovery period. The amplitudes (expressed in pV) are those obtained after averaging 128 stimulations. * P < 0.05; treated group versus control group.
the inhalation exposure and the electrophysiological measurements. The MCV and SCV of the tail nerve and the ASAP were adopted as parameters for testing peripheral nerve function in rats. Neurophysiological measurements were performed in a room maintained at 25-26“C, using a Racia-Medelec modular electrophysiological system equipped with a DAV 62 computer. The MCV was determined by eliciting a nerve impulse via electrical stimulation at two different sites of the tail and recording the action potential of the muscle innervated by the tail nerve. Electrical stimulation consisted of a 0.2-ms width impulse. The two stimulating electrodes were situated at 40 and
100 mm proximal to the recording electrode. The MCV was calculated by relating the distance between the two stimulating electrodes to the difference in the arrival time of the muscle action potential for the two sites of stimulation. This method allows the MCV to be measured independently of delay in the myoneural junction. For the determination of the SCV, the tip of the tail was stimulated and signals were recorded with the help of two electrodes situated at 40 and 100 mm proximal to the stimulating electrode. Stimulations, administered by a nerve train stimulator, consisted of 10 squarewave impulses per second, with an interpulse of 100 ms and a pulse width of 0.1 ms. The SCV was calculated
338
F. GAGNAJRE E T A L .
Table 1. Time-course of electrophysiological parameters during the recovery period in rats previously intoxicated with 500, 700 and 900 ppm DEB mixture" Electrophysiological parameter MCV (m s
l)
SCV Irn s-')
ASAPb (JLV)
Weeks of recovery 4
Peak
Group
0
2
I I1 Ill IV
Control 500 pprn 700 ppm 900 ppm
37.16 f 3.42 31.51 f 2.22" 30.54 f 2.04d 27.14 f 3.18",g,"
35.50 33.92 31.90 29.87
I II 111 IV
Control 500 ppm 700 pprn 900 ppm
43.46 f 3.57 40.76 f 2.74' 36.99 f 1.84d,f 33.89 f 1.92",gJ'
43.28 f 1.99 41.48 2 2.42 37.55 t 2.15',' 36.50 f 2.02=,g."
I
Control 500 ppm 700 ppm 900 pprn
208 2 174 5 181 2 153 2
236 32 177 2 31" 187 31d 152 f 40"
II
Ill IV
f 2.02
2.29 t 1.73',' f 2.62'.g." 2
*
25 24" 35d 23","
6
34.74 2 1.42 33.00 t 2.39 33.04 t 2.56 31.34 f 3.02'
36.05 33.24 33.45 31.96
t 3.10 2 2.29" t 1.28' t 2.42'
42.65 42.00 39.87 37.86
t 2.37 t 1.97 t 2.74'.' t 2.35".g
45.48 t 3.64 40.70 t 2.53' 40.15 f 2.36' 37.18 t 3.48",g,h
226 2 186 2 187 2 155 2
22 21" 35d 40e.8.h
232 ? 193 2 190 2 164 ?
32 21' 31' 33e,Q.h
The MCV, SCV and ASAP were obtained at the 18th week of exposure (week 0) and at the 2nd, 4th and 6th weeks of the recovery period. bThe amplitudes are those obtained after averaging 128 stimulations. Values are means f SD for 10 rats (controls) or 12 rats (exposed groups); P < 0.05. Group II versus Group I. Group Ill versus Group I. " Group IV versus Group I. Group 111 versus Group 11. Group IV versus Group II. Group IV versus Group 111. a
Q
Table 2. Latencies (in ms) of the five waveforms of BAEP at the 18th week of exposure to DEB mixture and at the 7th week of the recovery period P1
Group
18th week of exposure
7th week 18th of recovery week of period exposure
P2
P3
7th week 18th 7th week 18th of recovery week of of recovery week of period exposure period exposure
P4 7th week 18th of recovery week of period exposure
P5 7th week of recovery period
Control 1.17 (0.01) 1.14 (0.01) 1.70 (0.03) 1.67 (0.02) 2.42 (0.02) 2.44 (0.02) 3.08 (0.03) 3.06 (0.03) 4.49 (0.03) 4.45 (0.04) 600 pprn 1.22b (0.01) 1.21 (0.01) 1.81 (0.03) 1.77b (0.04) 2.55 (0.01) 2.54b (0.03) 3.23 (0.02) 3.24b (0.03) 4.62b (0.02) 4.66b (0.03) 800 ppm 1.26" (0.02) 1.24" (0.01) 1.87 (0.02) 1.79' (0.03) 2.69 (0.08) 2.68C.d(0.05) 3.28 (0.03) 3.25" (0.02) 4.91" (0.09) 4.75' (0.03) a
Values are means 2 SE. P < 0.05; exposed group versus control group. P < 0.05; 800 ppm-exposed group versus 600 ppm-exposed group.
b.c
by relating the distance between the two recording electrodes to the difference in the arrival time of the two averaged action potentials for the stimulus site. For the determination of the ASAP, the signal obtained by averaging 128 action potentials was recorded using the recording electrode situated in the middle part of the tail, and the amplitude was measured. During BAEP recordings, the test animal was held in a Plexiglas restraining device housed in an acoustically shielded chamber. The ambient temperature in the chamber was 23°C. Clicks of 50 ps duration (100 dB Sound Pressure Level) were used to elicit BAEP. The alternating polarity clicks were produced by applying square waves from a Racia ISD 45 stimulator to a JBL compression driver 2402 tweeter mounted 16.5 cm from the left pinea of the animal. Brainstem auditory responses were recorded with a
bandpass of 160 Hz to 3.2 kHz. The averaged response was recorded after 1028 clicks. The stimulus rate was 10 clicks s-' and evoked activity was analysed for 10 ms following each click. An artifact rejection system automatically suspended BAEP-averaging during movement. Before and after the recording sessions, the core temperature was monitored by a rectal probe (Ellab RM4). A heating lamp was used to maintain temperatures above 37°C when necessary. Each averaged response was plotted, and the latencies were measured from the onset of the acoustic signal to the positive peak of each wave, taking into account the transmission time of the signal between the loudspeaker and the ear of the animal. The central conduction time was calculated by subtracting the latency of the first component from that of the fifth component. Peak-topeak amplitudes were calculated from peaks N,P,.
339
INHALATION EXPOSURE TO DIETHYLBENZENE ma a
5T
a
4.5
c UJ +I
‘5 0
8
E
>
Y
P
4
4
t
1
0
3
5
7 9 12 Duration of exposure (weeks)
15
18
22
25
Recovery period (weeks)
Figure 3. Effect of DEB on the latencies of the first and fifth components of the BAEP. (0)Control group; DEB-exposed groups: (0) 600 ppm, (A)800 pprn. The arrow indicates the beginning of the recovery periods. Week 0 values are before exposure. a P < 0.05; exposed groups versus control group. P < 0.05; 800 pprn-exposed group versus 600 ppm-exposed group.
Statistical analysis
Statistical differences among groups were evaluated for each variable on each session by one-way analysis of variance, F values greater than those associated with the critical value at P < 0.05 were accepted as significant. Post hoc individual mean comparisons were made with Duncan’s multiple range test.4 The level of significance was set at P < 0.05. RESULTS General conditions Experiment A. Rats in the low, middle and high concentration groups were exposed to mean concentrations (5 SD) of 496 ? 45 ppm, 680 -1- 55 ppm and 869 2 96 ppm, respectively, i.e. 29, 40 and 51 ppm 1,2-DEB. As seen in Fig. IA, exposure to DEB reduced weight gain from the first week of exposure in each group.
There was no mortality in control, low and middle concentration groups. In the high concentration group, one animal was sacrificed on the 5th week of exposure because of an abscess at the neck. The animals of the middle and high concentration groups were prostrate during the exposure periods but recovered a few hours after the end of exposure. Rats in the t5ree exposed groups developed blue skin discoloration after 3 weeks of exposure. No animal in any group exhibited disturbances in gait or other signs of neurotoxicity. Experiment B . , Rats in the two exposure groups were exposed to mean concentrations (t SD) of 646 +- 42 ppm and 834 k 74 ppm, respectively, i.e. 38 and 49 ppm 1,2-DEB. As seen in Fig. lB, growth was retarded proportionally to the concentration of DEB mixture. From the 3rd week of exposure, the two exposed groups exhibited the blue skin discoloration. At the end of the exposure period, some animals presented disturbances in gait and one animal in the high exposure group had partially paralysed hindlimbs. In this latter group, two animals died during the
340
F. GAGNAIRE ET A L . 4 -
a b
a
12
15
b
3.5
3 0
3
5
7
9
18
22
25
Durationof exposure (was)
Figure4. Effect of DEB on the conduction time between the first and fifth components. (0)Control group; DEB-exposed groups: (0) 600 ppm, (A)800 ppm. The arrow indicates the beginning of the recovery period. Week 0 values are before exposure. P < 0.05; exposed groups versus control group. P < 0.05; 800 ppm-exposed group versus 600 ppm-exposed group.
4.5
3
2.5 0
5
7
9
Duration of exposure (weeks)
12
15
18
22
25
Recovery period (weeks)
Figure 5. Effect of DEB on the amplitude of the second component N,P, of the BAEP. (0)Control group; DEB-exposed groups: (0) 600 ppm, (A)800 pprn. The arrow indicates the beginning of the recovery period. Week 0 values are before exposure. P < 0.05; 800 ppm-exposed group versus control group.
INHALATION EXPOSURE TO DIETHYLBENZENE
exposure period. Moreover, many animals had to be sacrificed because they lost their head plugs during the recording sessions.
34 1
amplitude of the N,P, component was observed only on the high exposure group and although the deficit was statistically significant only at the 15th week of the exposure period (-30%), values were consistently lower through the experimental and recovery periods.
Neurophysiological examination Experiment A. Figure 2 summarizes the results of the conduction velocities and amplitudes of sensory action potentials in the tail nerve. During the entire exposure period, the MCV, SCV and ASAP increased in controls as well as in exposed animals. However, rats exposed to DEB exhibited deficits in the three electrophysiological parameters chosen as indicators of a neuropathy. In the 500 ppm DEBexposed group, only the ASAP differed significantly from control values. The MCVs were below control values but only at the 18th week of exposure and at the 6th week of the recovery period was the difference statistically significant. The SCVs did not differ from control values except at the 18th week of exposure and at the 6th week of the recovery period. In the 700 and 900 ppm-exposed groups, the three parameters statistically differed from control values. As seen in Fig. 2, the longer the exposure, the more pronounced the deficits were in the MCV, SCV and ASAP. Moreover, these effects were also concentration-dependent (cf. Table 1). The maximal deficits were reached at the 18th week of exposure. Electrophysiological values for control, low, middle and high concentrationexposed groups were, respectively (means .t SD): 37.16 t 3.42, 31.51 2 2.22 (-15yo), 30.54 t 2.04 (-18%)and27.14 t 3.18 ms-I (-27%)fortheMCV, 43.46 t 3.57, 40.76 -ir 2.74 (-6%), 36.99 5 1.84 (-15%) and 33.89 & 1.92 m s-I (-22%) for the SCV and 208 2 25, 174 -+ 24 (-l6%), 181 -+ 35 (-13%) and 153 t 23 FV (-26%) for the ASAP. During the recovery period, deficits in the MCV and SCV decreased partially, but at the 6th week the MCV and SCV values of the three exposed groups were always statistically different from control values (cf. Table 1). The ASAP showed no improvement in any DEB-exposed group; at the 6th week of the recovery period, the ASAP values for control, low, middle and high concentration-exposed groups were, respectively (means t SD): 232 t 32, 193 t 22 (-17*/0), 190 k 31 (-18%) and 164 2 33 FV (-29%). Experiment B. The BAEP consisted of a series of five principal waves consistently and reliably identified as previously described.' Figures 3, 4 and 5 show the absolute latencies of waves I and V, the I-V interpeak latencies and the amplitudes of the NzPl component, respectively. There was a concentration- and time-dependent increase in the latencies of the five components. The longer the treatment lasted, the more pronounced was the increase in the latencies. The fifth component was more affected than the first one, as shown by the increase of the I-V interpeak latency corresponding to the brainstem transmission time (Fig. 4). During the recovery period, the absolute latencies (Table 2) and the I-V interpeak latencies moved towards the control values but remained statistically different even after the 7th week of recovery. However, the effect on the
DISCUSSION
This study was undertaken with the purpose of examining the evidence for the occurrence of adverse effects on the peripheral and central nervous systems of rats chronically intoxicated by inhalation of DEB mixture. In accordance with our previous experiments,' we found that DEB caused blue discoloration in skin and urine. However, contrary to experiments with oral gavage,'.' we observed moderate clinical signs of neurotoxicity only in Experiment B. As atmospheric concentrations (646 and 834 ppm) were approximately the same as in Experiment A (680 and 869 ppm), this discrepancy may be explained by the difference in the age of animals in the two experiments. The intracellular protective mechanisms, which presumably deteriorate with increasing age, may be responsible for the development of hypersensitivity in 19-week-old rats (Experiment B) as compared to 9-week-old rats (Experiment A). Although no clinical sign of neurotoxicity was observed in Experiment A , the deficits in MCV and SCV were pronounced in the two high-exposure groups and the deficits in ASAP were clearly observed in all exposed groups. The lack of clinical signs of neurotoxicity in the presence of abnormal electrophysiological results has already been reported for other neurotoxicants, such as n-hexane and its or carbon disulphide.l(bl' A clear increase in the severity of changes in nerve conduction velocities and in the ASAP occurs with increasing exposure time. This relation between the duration of exposure to DEB and the extent of neurological abnormalities gives support to a causal connection between long-term exposure to DEB and impairment in peripheral nerve function. The relatively slow development of observed changes and their lack of reversibility exclude an acute effect, such as a membrane stabilizing action. Moreover, the higher the concentration of DEB, the more pronounced are the deficits in the electrophysiological parameters. The same phenomenon was observed with the increase in the absolute and interpeak latencies of BAEP. However, the action of DEB mixture by inhalation on the amplitudes of BAEP was ambiguous, contrary to that previously observed in rats receiving I,2-DEB by oral gavage in which the decreases of amplitudes of BAEP were pronounced.' Finally, it is assumed that the peripheral and central nervous functions of rats are altered by an l8-week inhalation exposure to 500 or 600 ppm DEB mixture vapours representing 30 or 36 ppm of the active isomer 1,2-DEB. These concentrations of 1,2-DEB are low compared to those of other neurotoxicants, such as nhexane or carbone disulphide, which need concentrations of 400 to 2000 ppm for producing neuropathies in 10.13-15
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F. GAGNAIRE E T A L
REFERENCES
1. F. Gagnaire, B. Marignac and J. De Ceaurriz, Diethylbenzeneinduced sensorimotor neuropathy in rats. J. Appl. Toxicol. 10, 105-112 (1990). 2. E. E. Sandmeyer, Aromatic hydrocarbons. In Patty’s Industrial Hygiene and Toxicology, 3rd Edn, ed. by G. D. Clayton and F. E. Clayton, pp. 3253-3431. Wiley, New York 11981). 3. F. Gagnaire, M. N. Becker and J. De Ceaurriz, Alteration of the brain stem auditory evoked potentials in diethylbenzene and diacetylbenzene-treated rats. J. Appl. Toxicol. 12 (51, 343-350 (1992). 4. S. Gad and C. S. Weil, Statistics and Experimental Design for Toxicologists, pp. 7G92. Telford Press, Caldwell, NJ ( 1986). 5. Y. Takeuchi, Y. Ono, N. Hisanaga, J. Kitoh and Y. Sigiura, A comparative study on the neurotoxicity of n-pentane, nhexane and n-heptane in the rat. Br. J. Ind. Med. 37, 241-247 (1980). 6. L. Perbellini, D. De Grandis, F. Semenzato, N. Rizzuto and A. Simonati, An experimental study on the neurotoxicity of n-hexane metabolites: hexanol-1 and hexanol-2. Toxicol. Appl. Pharmacol. 46,421-427 (1978). 7. R. J. Anderson and C. 6. Dunham, Electrophysiologic deficits in peripheral nerves as a discriminator of early hexacarbon neurotoxicity. J. Toxicol. Environ. Health 13, 835-843 (1984). 8. P. S. Spencer, H. H. Schaumburg, M. J. Sabri and B. Veronesi, The enlarging view of hexacarbon neurotoxicity.
CRC Crit. Rev. Toxicol. 7, 278-356 (1980). 9. S. Duckett, L. J. Streletz, R. A. Chambers, M. Auroux and P. Galle, 50 ppm MnBk subclinical neuropathy in rats. Experientia 35, 1365-1367 (1979). 10. F. Gagnaire, P. Simon, P. Bonnet and J. de Ceaurriz, The influence of simultaneous exposure to carbon disulfide and hydrogen sulfide on the peripheral nerve toxicity and metabolism of carbon disulfide in rats. Toxicol. Lett. 34, 175-183 (1986). 11. K. Knobloch, J. Stetkiewcz and T. Wronska-Nofer, Conduction velocity in the peripheral nerves of rats with chronic carbon disulphide neuropathy. Br. J. Ind. Med. 36, 148-152 (1979). 12. A. M. Seppalainen and I. Linnoila, Electrophysiological studies on rabbitsin long-term exposure to carbon disulfide. Scand. J. Work Environ. Health 1, 178- 183 ( 1975). 13. C. S. Rebert and E. Becker, Effects of inhaled carbon disulfide on sensory-evoked potentials of Long-Evans rats. Neurobehav. Toxicol. Teratol. 8, 533-541 (1986). 14. A. Colornbi, M. Maroni, 0. Picchi, E. Rota, P. Castano and V. Foa, Carbon disulfide neuropathy in rats. A morphological and ultrastructural study of degeneration and regeneration. Clin. Toxicol. 18, 1463-1474 (1981). 15. M. R. Gottfried, D. G. Graham, M. Morgan, H. W. Casey and J. S. Bus, The morphology of carbon disulfide neurotoxicity. Neurotoxicology 6, 89-96 (1985).
Diethylbenzene Inhalation-induced Electrophysiological Deficits in Peripheral Nerves and Changes in Brainstem Auditory Evoked Potentials in Rats F. Gagnaire,? M. N. Becker, B. Marignac, P. Bonnet and J. De Ceaurriz Institut National de Recherche et de Securite. 54501 Vandoeuvre, France
Key words: diethylbenzene; neurotoxicity; brainstem auditory evoked potentials; nerve conduction velocity; rats; inhalation exposure.
Motor and sensory conduction velocities (MCV and SCV), amplitude of the sensory action potential (ASAP) of the tail nerve and parameters of brainstem auditory evoked potentials (BAEP) were studied in male Sprague-Dawley rats after prolonged inhalation exposure to a commercial isomer mixture of diethylbenzene (DEB mixture) containing 6% 1,2-DEB. The MCV, SCV and ASAP were studied in one control group (10 rats) and three groups of 12 rats exposed to 500, 700 or 900 ppm DEB mixture for 6 h daily, 5 days per week, for 18 weeks. Rats used for recording BAEP (one control group and two other groups of 15 rats) were exposed to 600 and 800 ppm DEB mixture. The exposure time was the same. Rats exposed to DEB mixture exhibited a time- and concentration-dependentdecrease in MCV, SCV and ASAP and a time- and concentration-dependentincrease of both the peak latencies of all BAEP components and the interpeak (I-V) differences.
INTRODUCTION
MATERIALS AND METHODS
It was recently shown' that the commercial isomer mixture of diethylbenzene (DEB mixture), an intermediate of divinylbenzene synthesis,' administered to rats by oral gavage (750 or 500 mg kgg' once daily, 5 days per week, for 10 weeks) produced a timedependent decrease in motor and sensory conduction velocities (MCV and SCV) and in the amplitude of the sensory action potential (ASAP) of tail nerve. Rats treated with DEB mixture exhibited a ' blue discoloration of tissues and urine and presented evidence of clinical neuropathy leading to weakness of hind limbs and then paralysis. Oral administration of each separate isomer indicated that 1.2-DEB was the isomer responsible for neurotoxicity. Moreover, oral administration of 1.2-DEB produced a time-dependent increase of both the peak latencies of all brainstem auditory evoked potentials components (BAEP) and interpeak (I-V) differences and a decrease in the amplitude of the waveform.3 To our knowledge, the effects of long-term exposure have not been studied. The purpose of this study was to determine whether the same DEB mixture could cause peripheral and central sensorimotor neuropathies in rats when it was administered by inhalation at the three concentrations of 500, 700 and 900 ppm for a prolonged period of time. The integrity of the peripheral nervous system was investigated by measuring MCV, SCV and ASAP on the tail nerve and the integrity of the central nervous system was studied by using the brainstem auditory evoked potential (BAEP) technique.
Materials
t Author t o whom correspondence should be addressed. 026~437Xi921050335-OX$09.00 Wiley &L Sons. Ltd.
0 1991 by John
Diethylbenzene mixture (containing ca. 6% of 1,2DEB, 66% of 1,3-DEB and 28% of 1,4-DEB) was supplied by Aldrich Chemie, Steinheim, Germany. Animals
Ninety-one adult male Sprague-Dawley rats (IFFA Credo, Domaine des Oncins, Saint-Germain-sur1'Arbresle) were divided into two experimental groups A and B. Food (UAR-Alimentation, Villemoisson) and tapwater were available ad libitum. The rats were maintained under controlled environmental conditions with a 12-h dark/light cycle. Experimental design
In experiment A, 46 9-week-old rats were assigned to three exposed (12 rats each) and one control (10 rats) groups. The experimental groups were exposed to 500, 700 and 900 ppm DEB mixture for 6 h daily, 5 days per week, for 18 weeks. The control group was exposed to clean filtered air. The MCV, SCV and ASAP were measured every 2 weeks. At the end of the exposure period, the animals were kept for observation and neurophysiological measurements during a 6-week recovery period. In experiment B, 45 19-week-old rats were assigned to two exposed and one control group of 15 rats each. The two experimental groups were exposed to 600 or 800 ppm DEB mixture for 6 h daily, 5 days per week, Receiid 11 Ocrober I991 Acrepled (reimised) I N Fehruury 1992
336
F. GAGNAIRE ET A L . B
P
"1
700
650
600
550
500
450 0 1 2 3 4 5 6 7 8 9 10 11 11 13 14 15 16 17 I8 19 20 21 22 23 24
0 1 2 3 4 5 8 7 8 910111213141S1017181920212223242S
Figure 1. Effect of DEB on body weight. (A) Rats were exposed to 500 (O), 700 (0) or 900 (A)pprn of a mixture of three isomers of DEB for 6 h daily, 5 days per week. (B) Rats were exposed to 600 (0) or 800 (A)pprn of a mixture of three isomers of DEB for 6 h daily, 5 days per week. The control groups were exposed to clean filtered air (0).In parentheses: number of ratr alive (see text). The arrows indicate the beginning of the recovery period. Week 0 values are before exposure.
for 18 weeks. The control group was concurrently exposed to clean filtered air. The BAEP were recorded every 2 weeks during the first 9 weeks of exposure and every 3 weeks after. The survivors were kept for observation and neurophysiological measurements during a 7-week recovery period. Generation of test atmospheres
Exposures were conducted in 200-1 stainless-steel inhalation chambers with adjustable airflow (1C30 m3 '-I)' To prevent leakage, were maintained at negative pressure (2-3 mmH,O). An additional airflow was through the DEB mixture and Output vapour was with air to the required concentration before entering the exposure chamber. Atmosphere sampling and analysis
The levels of exposure to DEB were controlled by periodically pumping a measured volume of the test atmosphere through a glass tube packed with active charcoal. The DEB was desorbed from the activated charcoal with carbon disulphide. The resulting samples were then analysed for DEB using a 1 mx118 in. column filled wiih chromosorb W impregnated to 15% with 20 M polyethyleneglycol and heated to 90°C. The proportion of each isomer in the atmosphere samples was checked every 2 weeks for each concentration. The analytical procedure required the use of a 6 m x U 8 in. column filled with chromosorb W
impregnated to 20% with tris-cyanoethoxypropane and heated to 100°C. The proportions of the three isomers were 5.9 ? 0.4, 66.1 2 1.1 and 27.9 2 0.9% for 1,2-, 1,3- and 1,4-DEB, respectively. Surgical procedures
Implantation of electrodes was carried out under sodium pentobarbital anaesthesia (30 mg kg-', i.p.); xylazine (4 mg kg-, , i.p.) was used to tranquilize the animals and reduce the dose of pentobarbital. Electrodes consisted of three stainless-steel Screws (1.2 mmx2.5 mm); The reference electrode was placed 3 mm posterior to the lambda. The ground electrode was;placed over the nasal bone 2 mm anterior to the nasal suture and mm to the midline on the right. The active electrode was placed 2 mm posterior to bregma and 6 mm to the right of the midline. The screws were connected to a microconnector (Connectral) and the apparatus was cemented to the skull with dental acrylic. Each rat was given 50 mg of ampicillin (Totapen) and allowed to recover from the effects of surgery for at least 7 days before starting electrophysiological recordings. Neurophysiological measurements
The animals were kept at least 16 h in fresh air before the electrophysiological measurements, except after weekends when 48 h elapsed between the end of
337
INHALATION EXPOSURE T O DIETHYLBENZENE
40
A
T
35
30 Y
>
Y
25 I
0 1
3
5
7 10 12 Duration of exposure (weeks)
14
16
18
1
20 22 24 Recovery period (weeks)
B T
25 0 1
3
5
7 10 12 Duration of exposure (weeks)
14
16
18 20 22 24 Recovery period (weeks) C
250
T
T
0 1
3
5
7
10 12 Duration of exposure (weeks)
14
16
1
18 20 22 24 Recovery period (weeks)
500 ppm, (0) 700 ppm, (A) Figure 2. Effects of DEB on the MCV, SCV and ASAP. (0)control group; DEB-exposed groups: (0) 900 ppm. Week 0 values are before treatment. The arrow indicates the beginning of the recovery period. The amplitudes (expressed in pV) are those obtained after averaging 128 stimulations. * P < 0.05; treated group versus control group.
the inhalation exposure and the electrophysiological measurements. The MCV and SCV of the tail nerve and the ASAP were adopted as parameters for testing peripheral nerve function in rats. Neurophysiological measurements were performed in a room maintained at 25-26“C, using a Racia-Medelec modular electrophysiological system equipped with a DAV 62 computer. The MCV was determined by eliciting a nerve impulse via electrical stimulation at two different sites of the tail and recording the action potential of the muscle innervated by the tail nerve. Electrical stimulation consisted of a 0.2-ms width impulse. The two stimulating electrodes were situated at 40 and
100 mm proximal to the recording electrode. The MCV was calculated by relating the distance between the two stimulating electrodes to the difference in the arrival time of the muscle action potential for the two sites of stimulation. This method allows the MCV to be measured independently of delay in the myoneural junction. For the determination of the SCV, the tip of the tail was stimulated and signals were recorded with the help of two electrodes situated at 40 and 100 mm proximal to the stimulating electrode. Stimulations, administered by a nerve train stimulator, consisted of 10 squarewave impulses per second, with an interpulse of 100 ms and a pulse width of 0.1 ms. The SCV was calculated
338
F. GAGNAJRE E T A L .
Table 1. Time-course of electrophysiological parameters during the recovery period in rats previously intoxicated with 500, 700 and 900 ppm DEB mixture" Electrophysiological parameter MCV (m s
l)
SCV Irn s-')
ASAPb (JLV)
Weeks of recovery 4
Peak
Group
0
2
I I1 Ill IV
Control 500 pprn 700 ppm 900 ppm
37.16 f 3.42 31.51 f 2.22" 30.54 f 2.04d 27.14 f 3.18",g,"
35.50 33.92 31.90 29.87
I II 111 IV
Control 500 ppm 700 pprn 900 ppm
43.46 f 3.57 40.76 f 2.74' 36.99 f 1.84d,f 33.89 f 1.92",gJ'
43.28 f 1.99 41.48 2 2.42 37.55 t 2.15',' 36.50 f 2.02=,g."
I
Control 500 ppm 700 ppm 900 pprn
208 2 174 5 181 2 153 2
236 32 177 2 31" 187 31d 152 f 40"
II
Ill IV
f 2.02
2.29 t 1.73',' f 2.62'.g." 2
*
25 24" 35d 23","
6
34.74 2 1.42 33.00 t 2.39 33.04 t 2.56 31.34 f 3.02'
36.05 33.24 33.45 31.96
t 3.10 2 2.29" t 1.28' t 2.42'
42.65 42.00 39.87 37.86
t 2.37 t 1.97 t 2.74'.' t 2.35".g
45.48 t 3.64 40.70 t 2.53' 40.15 f 2.36' 37.18 t 3.48",g,h
226 2 186 2 187 2 155 2
22 21" 35d 40e.8.h
232 ? 193 2 190 2 164 ?
32 21' 31' 33e,Q.h
The MCV, SCV and ASAP were obtained at the 18th week of exposure (week 0) and at the 2nd, 4th and 6th weeks of the recovery period. bThe amplitudes are those obtained after averaging 128 stimulations. Values are means f SD for 10 rats (controls) or 12 rats (exposed groups); P < 0.05. Group II versus Group I. Group Ill versus Group I. " Group IV versus Group I. Group 111 versus Group 11. Group IV versus Group II. Group IV versus Group 111. a
Q
Table 2. Latencies (in ms) of the five waveforms of BAEP at the 18th week of exposure to DEB mixture and at the 7th week of the recovery period P1
Group
18th week of exposure
7th week 18th of recovery week of period exposure
P2
P3
7th week 18th 7th week 18th of recovery week of of recovery week of period exposure period exposure
P4 7th week 18th of recovery week of period exposure
P5 7th week of recovery period
Control 1.17 (0.01) 1.14 (0.01) 1.70 (0.03) 1.67 (0.02) 2.42 (0.02) 2.44 (0.02) 3.08 (0.03) 3.06 (0.03) 4.49 (0.03) 4.45 (0.04) 600 pprn 1.22b (0.01) 1.21 (0.01) 1.81 (0.03) 1.77b (0.04) 2.55 (0.01) 2.54b (0.03) 3.23 (0.02) 3.24b (0.03) 4.62b (0.02) 4.66b (0.03) 800 ppm 1.26" (0.02) 1.24" (0.01) 1.87 (0.02) 1.79' (0.03) 2.69 (0.08) 2.68C.d(0.05) 3.28 (0.03) 3.25" (0.02) 4.91" (0.09) 4.75' (0.03) a
Values are means 2 SE. P < 0.05; exposed group versus control group. P < 0.05; 800 ppm-exposed group versus 600 ppm-exposed group.
b.c
by relating the distance between the two recording electrodes to the difference in the arrival time of the two averaged action potentials for the stimulus site. For the determination of the ASAP, the signal obtained by averaging 128 action potentials was recorded using the recording electrode situated in the middle part of the tail, and the amplitude was measured. During BAEP recordings, the test animal was held in a Plexiglas restraining device housed in an acoustically shielded chamber. The ambient temperature in the chamber was 23°C. Clicks of 50 ps duration (100 dB Sound Pressure Level) were used to elicit BAEP. The alternating polarity clicks were produced by applying square waves from a Racia ISD 45 stimulator to a JBL compression driver 2402 tweeter mounted 16.5 cm from the left pinea of the animal. Brainstem auditory responses were recorded with a
bandpass of 160 Hz to 3.2 kHz. The averaged response was recorded after 1028 clicks. The stimulus rate was 10 clicks s-' and evoked activity was analysed for 10 ms following each click. An artifact rejection system automatically suspended BAEP-averaging during movement. Before and after the recording sessions, the core temperature was monitored by a rectal probe (Ellab RM4). A heating lamp was used to maintain temperatures above 37°C when necessary. Each averaged response was plotted, and the latencies were measured from the onset of the acoustic signal to the positive peak of each wave, taking into account the transmission time of the signal between the loudspeaker and the ear of the animal. The central conduction time was calculated by subtracting the latency of the first component from that of the fifth component. Peak-topeak amplitudes were calculated from peaks N,P,.
339
INHALATION EXPOSURE TO DIETHYLBENZENE ma a
5T
a
4.5
c UJ +I
‘5 0
8
E
>
Y
P
4
4
t
1
0
3
5
7 9 12 Duration of exposure (weeks)
15
18
22
25
Recovery period (weeks)
Figure 3. Effect of DEB on the latencies of the first and fifth components of the BAEP. (0)Control group; DEB-exposed groups: (0) 600 ppm, (A)800 pprn. The arrow indicates the beginning of the recovery periods. Week 0 values are before exposure. a P < 0.05; exposed groups versus control group. P < 0.05; 800 pprn-exposed group versus 600 ppm-exposed group.
Statistical analysis
Statistical differences among groups were evaluated for each variable on each session by one-way analysis of variance, F values greater than those associated with the critical value at P < 0.05 were accepted as significant. Post hoc individual mean comparisons were made with Duncan’s multiple range test.4 The level of significance was set at P < 0.05. RESULTS General conditions Experiment A. Rats in the low, middle and high concentration groups were exposed to mean concentrations (5 SD) of 496 ? 45 ppm, 680 -1- 55 ppm and 869 2 96 ppm, respectively, i.e. 29, 40 and 51 ppm 1,2-DEB. As seen in Fig. IA, exposure to DEB reduced weight gain from the first week of exposure in each group.
There was no mortality in control, low and middle concentration groups. In the high concentration group, one animal was sacrificed on the 5th week of exposure because of an abscess at the neck. The animals of the middle and high concentration groups were prostrate during the exposure periods but recovered a few hours after the end of exposure. Rats in the t5ree exposed groups developed blue skin discoloration after 3 weeks of exposure. No animal in any group exhibited disturbances in gait or other signs of neurotoxicity. Experiment B . , Rats in the two exposure groups were exposed to mean concentrations (t SD) of 646 +- 42 ppm and 834 k 74 ppm, respectively, i.e. 38 and 49 ppm 1,2-DEB. As seen in Fig. lB, growth was retarded proportionally to the concentration of DEB mixture. From the 3rd week of exposure, the two exposed groups exhibited the blue skin discoloration. At the end of the exposure period, some animals presented disturbances in gait and one animal in the high exposure group had partially paralysed hindlimbs. In this latter group, two animals died during the
340
F. GAGNAIRE ET A L . 4 -
a b
a
12
15
b
3.5
3 0
3
5
7
9
18
22
25
Durationof exposure (was)
Figure4. Effect of DEB on the conduction time between the first and fifth components. (0)Control group; DEB-exposed groups: (0) 600 ppm, (A)800 ppm. The arrow indicates the beginning of the recovery period. Week 0 values are before exposure. P < 0.05; exposed groups versus control group. P < 0.05; 800 ppm-exposed group versus 600 ppm-exposed group.
4.5
3
2.5 0
5
7
9
Duration of exposure (weeks)
12
15
18
22
25
Recovery period (weeks)
Figure 5. Effect of DEB on the amplitude of the second component N,P, of the BAEP. (0)Control group; DEB-exposed groups: (0) 600 ppm, (A)800 pprn. The arrow indicates the beginning of the recovery period. Week 0 values are before exposure. P < 0.05; 800 ppm-exposed group versus control group.
INHALATION EXPOSURE TO DIETHYLBENZENE
exposure period. Moreover, many animals had to be sacrificed because they lost their head plugs during the recording sessions.
34 1
amplitude of the N,P, component was observed only on the high exposure group and although the deficit was statistically significant only at the 15th week of the exposure period (-30%), values were consistently lower through the experimental and recovery periods.
Neurophysiological examination Experiment A. Figure 2 summarizes the results of the conduction velocities and amplitudes of sensory action potentials in the tail nerve. During the entire exposure period, the MCV, SCV and ASAP increased in controls as well as in exposed animals. However, rats exposed to DEB exhibited deficits in the three electrophysiological parameters chosen as indicators of a neuropathy. In the 500 ppm DEBexposed group, only the ASAP differed significantly from control values. The MCVs were below control values but only at the 18th week of exposure and at the 6th week of the recovery period was the difference statistically significant. The SCVs did not differ from control values except at the 18th week of exposure and at the 6th week of the recovery period. In the 700 and 900 ppm-exposed groups, the three parameters statistically differed from control values. As seen in Fig. 2, the longer the exposure, the more pronounced the deficits were in the MCV, SCV and ASAP. Moreover, these effects were also concentration-dependent (cf. Table 1). The maximal deficits were reached at the 18th week of exposure. Electrophysiological values for control, low, middle and high concentrationexposed groups were, respectively (means .t SD): 37.16 t 3.42, 31.51 2 2.22 (-15yo), 30.54 t 2.04 (-18%)and27.14 t 3.18 ms-I (-27%)fortheMCV, 43.46 t 3.57, 40.76 -ir 2.74 (-6%), 36.99 5 1.84 (-15%) and 33.89 & 1.92 m s-I (-22%) for the SCV and 208 2 25, 174 -+ 24 (-l6%), 181 -+ 35 (-13%) and 153 t 23 FV (-26%) for the ASAP. During the recovery period, deficits in the MCV and SCV decreased partially, but at the 6th week the MCV and SCV values of the three exposed groups were always statistically different from control values (cf. Table 1). The ASAP showed no improvement in any DEB-exposed group; at the 6th week of the recovery period, the ASAP values for control, low, middle and high concentration-exposed groups were, respectively (means t SD): 232 t 32, 193 t 22 (-17*/0), 190 k 31 (-18%) and 164 2 33 FV (-29%). Experiment B. The BAEP consisted of a series of five principal waves consistently and reliably identified as previously described.' Figures 3, 4 and 5 show the absolute latencies of waves I and V, the I-V interpeak latencies and the amplitudes of the NzPl component, respectively. There was a concentration- and time-dependent increase in the latencies of the five components. The longer the treatment lasted, the more pronounced was the increase in the latencies. The fifth component was more affected than the first one, as shown by the increase of the I-V interpeak latency corresponding to the brainstem transmission time (Fig. 4). During the recovery period, the absolute latencies (Table 2) and the I-V interpeak latencies moved towards the control values but remained statistically different even after the 7th week of recovery. However, the effect on the
DISCUSSION
This study was undertaken with the purpose of examining the evidence for the occurrence of adverse effects on the peripheral and central nervous systems of rats chronically intoxicated by inhalation of DEB mixture. In accordance with our previous experiments,' we found that DEB caused blue discoloration in skin and urine. However, contrary to experiments with oral gavage,'.' we observed moderate clinical signs of neurotoxicity only in Experiment B. As atmospheric concentrations (646 and 834 ppm) were approximately the same as in Experiment A (680 and 869 ppm), this discrepancy may be explained by the difference in the age of animals in the two experiments. The intracellular protective mechanisms, which presumably deteriorate with increasing age, may be responsible for the development of hypersensitivity in 19-week-old rats (Experiment B) as compared to 9-week-old rats (Experiment A). Although no clinical sign of neurotoxicity was observed in Experiment A , the deficits in MCV and SCV were pronounced in the two high-exposure groups and the deficits in ASAP were clearly observed in all exposed groups. The lack of clinical signs of neurotoxicity in the presence of abnormal electrophysiological results has already been reported for other neurotoxicants, such as n-hexane and its or carbon disulphide.l(bl' A clear increase in the severity of changes in nerve conduction velocities and in the ASAP occurs with increasing exposure time. This relation between the duration of exposure to DEB and the extent of neurological abnormalities gives support to a causal connection between long-term exposure to DEB and impairment in peripheral nerve function. The relatively slow development of observed changes and their lack of reversibility exclude an acute effect, such as a membrane stabilizing action. Moreover, the higher the concentration of DEB, the more pronounced are the deficits in the electrophysiological parameters. The same phenomenon was observed with the increase in the absolute and interpeak latencies of BAEP. However, the action of DEB mixture by inhalation on the amplitudes of BAEP was ambiguous, contrary to that previously observed in rats receiving I,2-DEB by oral gavage in which the decreases of amplitudes of BAEP were pronounced.' Finally, it is assumed that the peripheral and central nervous functions of rats are altered by an l8-week inhalation exposure to 500 or 600 ppm DEB mixture vapours representing 30 or 36 ppm of the active isomer 1,2-DEB. These concentrations of 1,2-DEB are low compared to those of other neurotoxicants, such as nhexane or carbone disulphide, which need concentrations of 400 to 2000 ppm for producing neuropathies in 10.13-15
342
F. GAGNAIRE E T A L
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