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Toxicology and Applied Pharmacology 200 (2004) 131–145

Neurotoxicity of carbonyl sulfide in F344 rats following inhalation

exposure for up to 12 weeks

Daniel L. Morgan,a Peter B. Little,b David W. Herr,c Virginia C. Moser,c Bradley Collins,d

Ronald Herbert,e G. Allan Johnson,f Robert R. Maronpot,e G. Jean Harry,a and Robert C. Sillse,*

aLaboratory of Molecular Toxicology, NIEHS, Research Triangle Park, NC 27709, USAbPathology Associates Division of Charles River Laboratories, Durham, NC 27713, USA

cNeurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27511, USAdLaboratory of Pharmacology and Chemistry, NIEHS, Research Triangle Park, NC 27709, USA

eLaboratory of Experimental Pathology, NIEHS, Research Triangle Park, NC 27709, USAfDuke University Medical Center, Durham, NC 27710, USA

Received 20 January 2004; accepted 12 April 2004

Available online 20 July 2004

Abstract

Carbonyl sulfide (COS), a high-priority Clean Air Act chemical, was evaluated for neurotoxicity in short-term studies. F344 rats were

exposed to 75–600 ppm COS 6 h per day, 5 days per week for up to 12 weeks. In rats exposed to 500 or 600 ppm for up to 4 days, malacia

and microgliosis were detected in numerous neuroanatomical regions of the brain by conventional optical microscopy and magnetic

resonance microscopy (MRM). After a 2-week exposure to 400 ppm, rats were evaluated using a functional observational battery. Slight gait

abnormality was detected in 50% of the rats and hypotonia was present in all rats exposed to COS. Decreases in motor activity, and forelimb

and hindlimb grip strength were also detected. In rats exposed to 400 ppm for 12 weeks, predominant lesions were in the parietal cortex area

1 (necrosis) and posterior colliculus (neuronal loss, microgliosis, hemorrhage), and occasional necrosis was present in the putamen, thalamus,

and anterior olivary nucleus. Carbonyl sulfide specifically targeted the auditory system including the olivary nucleus, nucleus of the lateral

lemniscus, and posterior colliculus. Consistent with these findings were alterations in the amplitude of the brainstem auditory evoked

responses (BAER) for peaks N3, P4, N4, and N5 that represented changes in auditory transmission between the anterior olivary nucleus to the

medial geniculate nucleus in animals after exposure for 2 weeks to 400 ppm COS. A concentration-related decrease in cytochrome oxidase

activity was detected in the posterior colliculus and parietal cortex of exposed rats as early as 3 weeks. Cytochrome oxidase activity was

significantly decreased at COS concentrations that did not cause detectable lesions, suggesting that disruption of the mitochondrial

respiratory chain may precede these brain lesions. Our studies demonstrate that this environmental air contaminant has the potential to cause a

wide spectrum of brain lesions that are dependent on the degree and duration of exposure.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Carbonyl sulfide; Neurotoxicity; Magnetic resonance microscopy; Auditory system; Brainstem auditory evoked responses; Cytochrome oxidase

Introduction sources of COS include its use as an intermediate in the

Carbonyl sulfide (COS) is a by-product of coal hydroge-

nation and gasification, viscose rayon production (Houben-

Weyl, 1955), is a component of cigarette smoke (Wynder and

Hoffmann, 1967), and has been identified as a potentially

toxic component of air pollution. The largest man made

0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2004.04.013

* Corresponding author. Laboratory of Experimental Pathology,

National Institute of Environmental Health Sciences, 111 Alexander Drive,

South Campus, MD B3-08, P.O. Box 12233, Research Triangle Park, NC

27709. Fax: +1-919-541-4714.

E-mail address: sills@niehs.nih.gov (R.C. Sills).

production of thiocarbamate herbicides and pesticides (EPA,

1994). It is also released as a combustion by-product from

automobile exhaust and in the manufacture of petroleum and

rubber products (EPA, 1994). The atmospheric half-life of

COS is estimated to be 2 years. Carbonyl sulfide was placed

on the list of Clean Air Act Chemicals—Hazardous Air

Pollutants because of reported high emissions (9500 tons per

year). Carbonyl sulfide is a metabolite of carbon disulfide, a

known neurotoxicant (Beauchamp et al., 1983). Although

total COS environmental emissions are relatively high, there

is a lack of toxicity data for this chemical.

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145132

The major concern regarding COS toxicity is potential

neurotoxicity. In an earlier study (Monsanto, 1985),

Sprague–Dawley rats received a 4-h exposure to 1062–

1189 ppm COS. Central nervous system dysfunction was

reported 14 days after exposure. In another study (Nutt et

al., 1996), F344 rats exposed for 4 h to 750–1000 ppm COS

and held for 14 days had vacuolation of the myelin sheath in

the cerebellar peduncles, corpus collosum, and pyramidal

tracts, while rats exposed to 500–590 ppm COS and held

for 3–14 days had focal necrosis or gliosis of the cerebellar

cortex and peduncles.

Based on the limited neurotoxicological and neuropath-

ological evaluation in the peer-reviewed literature, COS was

selected for further toxicological evaluation in the current

study. The objectives of this study were to examine the

neurobehavioral effects of COS exposure and characterize

the cellular responses by light microscopy; evaluate the

neuroanatomical targets by magnetic resonance microscopy

(MRM); characterize the early electrophysiological and

biochemical effects of COS toxicity; and determine the

dose-response relationships of subchronic exposure to COS.

Methods

Range-finding study. Because of the paucity of exposure

data available for COS, a range-finding experiment was

conducted. Male F344 rats (5 per concentration) were

exposed to 0, 75, 150, 300, or 600 ppm COS, 6 h per day

for 4 days. In an attempt to replicate an earlier study (Nutt et

al., 1996), a second group of male rats (5 per concentration)

was exposed to 0, 75, 150, 300, or 600 ppm for 6 h and then

held for 2 weeks without exposure. Rats were anesthetized

(Nembutal, ip), cardiac perfused, and brains were removed

and processed for histological evaluation. Brains were

embedded in paraffin, sectioned at approximately 5 Am,

stained with hematoxylin and eosin (H&E), and evaluated

microscopically.

Two-week repeated exposure. Based upon initial range-

finding results, a 2-week experiment was conducted to

evaluate the effects of repeated exposure to 0, 300, 400,

or 500 ppm COS. Male and female rats (10 per sex per

concentration) were exposed for 6 h per day, 5 days per

week for 12 exposures. Neurobehavioral evaluations were

conducted using a functional observational battery (FOB)

and an automated assessment of motor activity. For the 2-

week study, rats were tested 2 days before COS exposure

(FOB and motor activity), immediately after the first expo-

sure (FOB only), and the morning following the last

exposure (FOB and motor activity). After conducting the

FOB, all rats were anesthetized (Nembutal, ip), cardiac

perfused, and tissues collected for histopathology evalua-

tion. Brains were embedded in paraffin, sectioned at ap-

proximately 5 Am, stained with H&E, and evaluated

microscopically.

Twelve-week exposure. Based upon the results of the 2-

week exposure, a 12-week study was performed at concen-

trations that were not acutely toxic. Male and female F344

rats (20 per sex per concentration) were exposed to 200,

300, or 400 ppm COS 6 h per day, 5 days per week for 12

weeks. Controls were exposed to filtered, conditioned air.

For the 12-week exposure, rats were tested with the FOB

(no motor activity) during the sixth week of exposure

(immediately following exposure) and the morning after

the last exposure. As before, rats were euthanized for

neuropathology after FOB testing. Additional groups of rats

were included for clinical pathology, electrophysiological

testing, magnetic resonance microscopy (MRM), and cyto-

chrome oxidase analyses (see below).

Chemical. Carbonyl sulfide (CAS# 463-58-1) was pur-

chased from Tex-La Gases (Houston, TX). Carbonyl sulfide

was procured as a liquid in gas cylinders equipped with

valves configured to provide the vapor phase of COS.

Chemical purity was determined to be >98.1% by gas

chromatography/thermal conductivity analysis of the vapor

phase over the liquid COS. The 1.9% residual was com-

posed primarily of CO2 with <0.6% H2S. After dilution of

the bulk gas to the desired COS exposure concentrations,

the H2S concentrations were reduced to insignificant levels.

No H2S could be detected in air samples collected from the

chambers during COS exposures.

Inhalation exposure. The COS vapor, at reduced pressure,

was supplied to three variable speed pumps that controlled

the vapor flow necessary to achieve the desired concentra-

tion for each exposure chamber. The COS vapor was mixed

with conditioned air (HEPA filtered, charcoal scrubbed,

temperature and humidity controlled) and delivered to the

Hazleton 2000 exposure chambers at approximately 400 L

min�1. The COS concentration in each chamber was sam-

pled at about 90-s intervals and measured using Orbital

Scientific Model Diamond 20 Fourier transform infrared

(FTIR) spectrophotometers. Exhaust from the exposure

chambers was passed through three activated charcoal

scrubbers (Safemod model CA-500) connected in series.

The scrubbed exhaust was sampled about every 15 min and

analyzed for COS. The concentrations of COS in the

exposure chambers were independently verified before and

during the animal exposures. Chamber samples were ana-

lyzed by gas chromatography (GC) with photo-ionization

and GC-thermal conductivity detectors.

Animals. Male and female Fischer 344 rats (Charles River

Laboratories, Raleigh, NC), 6–7 weeks old on arrival, were

held for 10–14 days to confirm absence of disease and to

acclimate to the exposure facility. During the holding period,

rats were weighed and randomized to treatment groups.

Animals were placed in exposure chambers without food 6

h per day for 2 days before chemical exposure for acclimation

to the exposure conditions. Animals were 8–9 weeks old at

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145 133

the start of the exposures. Animals were individually housed

in Hazleton 2000 inhalation exposure chambers for the

duration of the study. Feed (NIH-07) was removed during

the 6-h exposures and for 6 h per day on nonexposure days.

Water (chlorinated, city water) was provided ad libitum by an

automatic watering system during the nonexposure as well as

the exposure periods. Individual body weights were recorded

for all animals on the day before the first exposure andweekly

thereafter. Rats were observed for clinical signs of toxicity

two times daily (once in the early morning and once in the late

afternoon). Individual animal clinical observations were

documented for all rats at the time of weighing. Animals

were exposed to COS for 6 h per day (approximately 7 AM to

1 PM), 5 days per week (weekends excluded) for up to 12

weeks. All animals were exposed for two consecutive days

before termination. Control animals breathed filtered, condi-

tioned air.

This study was conducted under federal guidelines for

the use and care of laboratory animals and was approved by

the NIEHS Animal Care and Use Committee. Animals

were housed in a humidity- and temperature-controlled,

HEPA-filtered, mass air displacement room in facilities

accredited by the American Association for Accreditation

of Laboratory Animal Care. Animal rooms were maintained

with a light–dark cycle of 12 h (light from 0700 to 1900

h). Sentinel animals, housed in the animal facility as part of

an ongoing surveillance program for parasitic, bacterial,

and viral infections, were pathogen-free throughout the

study.

Functional observational battery (FOB). Behavioral and

neurological changes were assessed using the FOB and an

evaluation of motor activity. The FOB protocol used in this

study was based on previously reported procedural details

and scoring criteria (McDaniel and Moser, 1993). Upon

removal from the cage, the observer ranked the rat’s

reactivity to being held, and in addition noted and ranked

any changes in general appearance, including lacrimation,

salivation, ptosis, pupil size, and piloerection. Next, the rat

was placed on top of a laboratory cart (open field) for 2

min of undisturbed observations of activity level, arousal,

posture, gait, and occurrence of involuntary motor move-

ments (e.g., tremors, convulsions). Next, a series of reflex

tests was conducted that consisted of ranking each rat’s

responses to an auditory click stimulus using a metal

clicker (click response), a pinch on the tail using forceps

(tail-pinch response), and the ability of the pupil to

constrict to a penlight stimulus (pupil response). Aerial

righting was assessed by ranking the ability of the rat to

land on all four feet when dropped from a supine position

30 cm above a padded surface. Forelimb and hindlimb grip

strength were quantified using strain gauges and the force

necessary to break the rat’s grip was recorded. Landing

foot splay, the distance between the hindpaws when

dropped from 30 cm, was conducted last. The same

observer conducted all tests and was blind with respect

to the concentration levels. Motor activity testing was

conducted using photocell-based chambers. The activity

system consists of frames made to fit around a standard

rat cage, with seven photocells evenly spaced across the

length of the cage to detect movement. The activity session

lasted for 30 min.

Clinical pathology. On the morning after the last exposure

(12 weeks), 5 rats per sex per concentration were anesthe-

tized (Nembutal, ip) and blood was collected by cardiac

puncture. Blood samples were centrifuged in serum collec-

tion vials at 500 � g for 10 min. Serum samples were

analyzed for alanine aminotransferase (ALT), alkaline phos-

phatase (AP), aspartate aminotransferase (AST), cholesterol,

sorbitol dehydrogenase (SDH), total protein, creatine kinase

(CK), creatinine, urea nitrogen (UN), and glucose using an

automated analyzer (Monarch System 2000, Instrumenta-

tion Laboratory, Lexington, MA) and commercially avail-

able reagents.

Histopathology. Immediately following FOB evaluations

at the end of the COS exposures, animals were anesthetized

(Nembutal, ip) and perfused via the left ventricle with an

initial flushing solution of 0.9% sodium chloride containing

1000 units/L heparin sodium and 1 ml/L of 1% sodium

nitrite (approximately 1 min) followed by McDowell–

Trump’s fixative (McDowell and Trump, 1976) at a rate

of approximately 30 ml/min for 10–15 min. Pressure was

by gravity from approximately 1 m above the table level.

The brain and other tissues were harvested and placed into

perfusate. Using ventral topographic markers, perpendicu-

lar transverse (coronal) sections of the brain were prepared

from the following six regions: (1) frontal cortex through

the chiasma, (2) frontoparietal cortex through the infundib-

ulum, (3) mid-anterior colliculi, (4) posterior colliculi at the

level just anterior to the pons, (5) cerebellum and medulla

at its midpoint through the cochlear nuclei, and (6) obex at

the posterior medulla at the origin of the spinal central

canal. Brain sections were embedded in paraffin, sectioned

at approximately 5 Am, and stained with hematoxylin and

eosin (H&E). All sections were evaluated by a board-

certified veterinary pathologist with expertise in evaluating

the central and peripheral nervous systems.

Brainstem auditory evoked potentials. A preliminary as-

sessment of electrophysiological changes resulting from the

neuropathology in the brainstem in a subset of animals in

the 12-week study was made after 2 weeks exposure to

COS. Groups (n = 10 per group) of male animals exposed to

0 or 400 ppm COS were removed from the chambers after

10 exposures for brainstem auditory evoked response

(BAER) testing. The BAER was used as a measure of the

auditory neural function of the rats. The BAER is an

electrophysiological response to auditory stimuli and repre-

sents neural transmission from the auditory nerve through

the level of the brainstem (Hall, 1992). Two days after the

Table 1

Incidence of neuropathological lesions in male rats exposed to 600 ppm

COS

CNS regiona Neuropathological Duration

lesionsControl 1 dayb 2 daysc

Brain lesions n = 5 n = 5 n = 5

Parietal cortex area 1 Cortical necrosis 0/5 0/5d 5/5*

Restrosplenial cortex Cortical necrosis 0/5 0/5 2/5

Putamen Necrosis 0/5 1/5 0/5

Internal capsule Necrosis 0/5 2/5 0/5

Thalamus Necrosis 0/5 2/5 5/5**

Pyriform cortex Necrosis 0/5 0/5 2/5

Red nucleus Vacuolation 0/5 0/5 2/5

Anterior

olivary nucleus

Vacuolation

and/or necrosis,

axonopathy

0/5 1/5 3/4*

Posterior colliculus Necrosis 0/5 0/5 5/5**

Cerebellar cortex Necrosis 0/5 1/4 0/5

Cerebellar

roof nucleus

Vacuolation

and/or hemorrhage

0/5 0/4 3/4*

Cerebellar

roof nucleus

Necrosis and

cavitation

0/5 3/3* 0/4

Cerebellar

medullary white

Vacuolation 0/5 5/5** 0/5

Fifth cranial

nerve tract

Vacuolation 0/5 5/5** 0/5

a Thirty-six specific-named areas of brain were recorded in the delta sheets

of six standard sections of brain.b Male rats exposed to COS for 1 day and then held of 14 days.c Male rat exposed to COS for 2 days and sacrificed.d Number of animals with the lesion/number of animals for which that area

of brain was examined.

*P < 0.05 vs. controls (Fisher’s exact test).

**P < 0.001 vs. controls (Fisher’s exact test).

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145134

10th exposure, animals were surgically implanted with

electrodes using procedures that have been described pre-

viously (Herr and Boyes, 1997; Herr et al., 1992, 1994).

Electrode locations were as follows: (1) the active electrode

for BAERs was 3 mm posterior to lambda on the midline,

(2) a ground electrode was 2 mm anterior to bregma and 2

mm to the left of midline, and (3) a reference electrode was

7 mm anterior to bregma and 2 mm to the right of midline.

The rats were allowed to recover approximately 9 days

before testing. Auditory stimuli were calibrated, generated,

presented, and the BAERs were recorded using the system

previously described (Hamm et al., 2000; Herr et al., 1996).

Each subject’s BAER waveform was the average of the

neural responses to 500 stimuli. The auditory stimuli con-

sisted of rarefaction clicks (50 As) at an intensity of

approximately 80 dBpeak SPL (re: 20 APa). The speaker

was placed approximately 10.7 cm in front of, and 26 cm

above, the animal’s auditory canals. This resulted in a

distance of about 28.1 cm between the speaker and the

auditory canals, at an angle of approximately 68j. The

acoustic travel time was approximately 839 As. Due to the

known influence of temperature on evoked potentials (de

Jesus et al., 1973; Hetzler and Dyer, 1984; Hetzler et al.,

1988; Janssen et al., 1991; Miyoshi and Goto, 1973;

Petajan, 1968), colonic temperature was quantified imme-

diately following the animal’s removal from the test cham-

ber. A temperature probe (Model RET-1; Physitemp

Instruments, Inc., Clifton, NJ), connected to a thermometer

(Model BAT-10, Physitemp Instruments, Inc.), was inserted

approximately 8 cm rectally and deep colonic temperature

was recorded. These data were used to ascertain if changes

in body temperature could be related to alterations in

evoked potentials.

Peak amplitudes and latencies were measured from each

animal’s average waveform. Peak amplitudes (in AV) weremeasured from baseline (defined as the average voltage over

the prestimulus period). Peak latencies (in ms) were calcu-

lated from stimulus onset. Peaks were identified by their

polarity and latency according to the average waveform

from each treatment group. Data were analyzed using an

analysis of variance (ANOVA; PROC GLM) (SAS Institute,

1989, 1997) with the concentration of COS treated as a

between-subject factor. A critical a level of V0.05 was used

for all analysis in this exploratory study. Data are reported as

mean F SE. Group-averaged waveforms were calculated

from individual animal data and are presented for illustrative

purposes.

Cytochrome C oxidase assay. In initial studies (Nutt et al.,

1996), there was some suggestion that COS may inhibit

mitochondrial enzymes. To further explore potential mech-

anisms of COS neurotoxicity, the rate-limiting enzyme in

the mitochondrial respiratory chain (cytochrome c oxidase)

was evaluated in the current study. Our hypothesis is that

COS inhibits mitochondrial enzymes in the areas of the

brain with high energy requirements.

After exposure for 3, 6, and 12 weeks, 5 rats per sex per

exposure concentration were euthanized (Nembutal, ip) and

the brains rapidly removed and flash frozen in liquid nitro-

gen. Regions approximating the parietal cortex and posterior

colliculus were dissected from frozen brains. The reported

method (Dorman et al., 2002) was modified to prepare the

frozen brain sections for the cytochrome oxidase assay.

Portions (approximately 50 mg) of the posterior colliculus

and parietal cortex were homogenized in 15 ml of ice-cold 10

mM HEPES buffer (pH 7.4, 50 mM sucrose, 200 mM

mannitol, 1 mM EDTA). The homogenate was centrifuged

for 10 min at 3000� g at 4 jC. The pellet was discarded andthe supernatant used for the cytochrome oxidase assay.

Cytochrome oxidase activity was determined by the

reported method (Hess and Pope, 1963) and adapted for

use in a microplate reader (Bio-Tek, EL-340). A stock

solution of 0.25 mM cytochrome c was prepared in 50

mM HEPES buffer and stored for up to 1 week at 4 jC.Working concentrations of reduced cytochrome c (0.025

mM) were prepared daily in the same buffer and reduced

by adding sodium hydrosulfite up to 1 AM. Excess hydro-

sulfite was oxidized by shaking the solution for 3 min. The

degree of reduction was evaluated by measuring the ratio of

absorbance (Beckman DU640 spectrophotometer) at 550–

Fig. 1. Male F344 rats were exposed to 600 ppm carbonyl sulfide (COS) for 1 day (6 h) and held for 2weeks. Themagnification of all figures represents the original

magnification (objective � zoom lens) at the time of photomicroscopy. (A) Bilateral symmetrical areas of necrosis and microgliosis in the internal capsule

(arrows), H&E, 3.3�. (B) Focal necrosis with cavitation of the cerebellar white matter, granular layer, andmolecular layer (arrow), H&E, 8�. (C)Multifocal areas

of necrosis and gliosis in cerebellar roof nuclei (arrows), H&E, 2.5�. (D) Gliosis is characterized by accumulations of microglial cells and macrophages within an

area of malacia, H&E, 40�.

Fig. 2. Male F344 rats were exposed to 600 ppm carbonyl sulfide (COS) for 2 days (6 h per day). (A) Note the predominant bilateral symmetrical necrosis in area 1

of the parietal cortex (arrow head), in the thalamus (arrows) and within the retrosplenial granular cortex, H&E, 2.5�. (B) Similar bilateral symmetrical necrosis is

seen in the posterior colliculi (arrows), H&E, 2.5�. (C) Higher magnification of parietal cortex area 1. The extensive area of necrosis is characterized by

microvacuolation of the neuropil especially at the periphery (arrows) and the presence of mutifocal hemorrhage (arrowheads), H&E, 13.2�. (D) Compared to the

adjacent normal parietal cortex (N), neurons in the area of necrosis are shrunken and basophilic (arrows). The lesion is morphologically early in type, H&E, 50�.

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145 135

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145136

565 nm (Dorman et al., 2002). Working solutions prepared

by this method consistently yielded measured ratios greater

than 6.5. The supernatant was diluted 10-fold in 10 mM

HEPES buffer and 10 Al was added to 200 Al of reducedcytochrome c solution. Enzyme activity was measured by

following the decrease in absorbance at 550 nm every 15 s

for 30 measurements.

The total protein in each supernatant sample was mea-

sured by modifying the Bradford assay (Sigma, St. Louis,

MO) for use in the microplate reader. Bovine serum albumin

standards were prepared to encompass the range of 0.095–

1.5 mg/ml. Ten microliters of undiluted supernatant or

standard was added to 200 Al of Bradford reagent, incubated

at room temperature for 5 min, and the absorbance was read

at 595 nm.

Magnetic resonance microscopy (MRM). A preliminary

MRM study was conducted in parallel with the 12-week

study based on the findings (Nutt et al., 1996) that

indicated the brain was a potential target for COS neuro-

toxicity. The rationale for including MRM in the study is

that it is the imaging modality of choice for identifying

changes in neuroanatomical structures, and 200 brain slices

can be evaluated without destroying the brain. F344 rats

were exposed to 0 or 400 ppm COS during the 12-week

studies.

Before MR imaging, rats were anesthetized and cardiac

perfused with McDowell Trump’s fixative containing

gadolinium MR contrast agent. The brains were removed

and the fixed specimens were scanned at 9.4 T using spin

echo encoding (TR = 100 ms, TE = 3.2 ms) for 3.5 h.

The scans provided a 256 � 256 � 256 image array with

a resolution of 70 � 70 � 140 Am (7 � 10�4 mm3).

Details of MRM methods have been published previously

(Johnson et al., 2002). After the images were analyzed,

the fixed whole brains were placed in neutral-buffered

formalin. Abnormal findings identified in the MRM

images provided a guide for determining where brain

sections were going to be made for subsequent histolog-

ical evaluation (Sills et al., 2004).

Table 2

Body weight of F344 rats exposed to COS for 2 weeks

COS Males Females

(ppm)Numbers of exposure Numbers of exposure

0 5 12 0 5 12

0 222 F 12a 235 F10 252 F 11 155 F 3 160 F 4 164 F 6

300 219 F 15 229 F 11 248 F 11 160 F 2 163 F 4 168 F 3

400 221 F 14 231 F 14 243 F 16 158 F 5 160 F 2 162 F 4

500 218 F 15 202 F 14* b 157 F 3 145 F 9* 159 F 6c

a Values are in grams and represent means F SD, n = 10.b No survivors.c N = 6 rats.

*Significantly less than control P < 0.05.

Results

Range-finding experiment

Clinical observations

No mortality, morbidity, or clinical signs of toxicity

were observed in rats exposed to 75, 150, or 300 ppm

COS for 4 days. However, some rats exposed to 600 ppm

were euthanized in moribund condition after 2 days of

exposure. Animals exposed to 600 ppm exhibited clinical

signs of hypothermia, lethargy, ataxia, and impaired right-

ing reflex.

No mortality occurred in rats that received one 6-h expo-

sure to 75, 150, 300, or 600 ppm COS and were then held

for 2 weeks. Animals exposed to 600 ppm were lethargic

when observed immediately after exposure and the follow-

ing morning (day 2). By the afternoon of day 2, these rats

exhibited clinical signs of hypothermia, lethargy, head tilt,

and ataxia. These clinical signs were less severe than those

observed in rats receiving two exposures to 600 ppm. The

clinical conditions improved during the 14-day holding

period; however, several rats continued to exhibit ataxia

with head tilt.

Histopathology

No microscopic brain lesions were observed in rats

exposed to 75, 150, or 300 ppm COS for 4 days. Micro-

scopic evaluation of brain sections from rats exposed to 600

ppm COS for 1 day and held for 2 weeks included necrosis

and microgliosis in the cerebellar roof nucleus, internal

capsule, and thalamus (Table 1, Fig. 1). Also present was

vacuolation of the cerebellar medullary white matter and

fifth cranial nerve tract. Microscopic evaluation of brain

sections from moribund animals exposed to 600 ppm COS

for 2 days revealed extensive bilateral symmetrical necrosis

in parietal cortex area 1 and thalamus (Table 1, Fig. 2).

Necrosis was also observed in the retrosplenial granular

cortex, pyriform cortex, red nucleus, cerebellar roof nucleus,

posterior collicular nucleus, and anterior olivary nucleus.

Although the posterior colliculus is not typically examined

in standard brain survey sections, a fortuitous section in one

animal indicated severe necrosis. Based on this finding and

the consistent detection of hypointense areas (gliosis) in the

posterior colliculus by magnetic resonance microscopy

(MRM), in subsequent experiments, animals were examined

for midbrain lesions. No brain lesions were observed in

animals exposed to 75, 150, or 300 ppm COS for 6 h and

held for 2 weeks.

Two-week repeated exposure

Clinical observations

All male (10/10) and female (4/10) rats exposed to 500

ppm COS for 2 weeks were euthanized in moribund

condition and removed from the study. Male rats were

D.L. Morgan et al. / Toxicology and Applied

found moribund after 4 (1/10), 5 (6/10), and 10 (3/10)

exposures. Similarly, females were found moribund after 5

(2/10) and 11 (2/10) exposures. Moribund animals exhibited

clinical signs of hypothermia, lethargy, ataxia with poor

control of front and rear limbs. Rats exposed to 300 and 400

ppm COS exhibited no adverse clinical signs.

Body weights

Body weights of male and female rats receiving 12

exposures to 300 and 400 ppm COS were not significantly

different from controls (Table 2). Body weights of surviving

males and females in the 500 ppm exposure groups were

significantly less than controls (P<0.05) after five exposures.

Fig. 3. Brain from a female F344 rat exposed to (A) filtered air (control) or (B) 50

symmetrical malacia in the frontoparietal cortex (arrows). (C) In addition to the e

extensive malacia in the thalamus (arrows), H&E, 3.3�. (D) Necrosis of the retrosp

ppm COS, H&E, 13.2�.

Functional observational battery (FOB)

At 500 ppm, surviving females showed significantly

decreased grip strength (both forelimb and hindlimb), hypo-

tonia, and slight gait abnormalities. Similar but lesser effects

were seen at 400 ppm: slight gait changes in about half the

rats and hypotonia (both sexes). Both 300 and 400 ppm

produced slightly increased handling reactivity, but this effect

was transient and not clearly dose responsive. No changes in

activity levels or sensorimotor responses were detected.

Histopathology

Bilateral symmetrical malacia of the frontoparietal cor-

tex was observed upon gross examination of brains from

Pharmacology 200 (2004) 131–145 137

0 ppm COS for 5 days. Compared to the control, note the marked bilateral

xtensive malacia in the parietal cortex area 1 (arrowheads), note the locally

lenial cortex (arrows) was a common lesion observed in rats exposed to 500

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145138

rats exposed to 400 and 500 ppm COS for 2 weeks (Figs.

3B and 4A). Microscopic lesions were present in the brain

of all early death rats exposed to 500 ppm and in 8/10 male

and 9/10 female rats exposed to 400 ppm for 2 weeks

(Table 3). Brain lesions were present in one female exposed

to 300 ppm for 2 weeks. Predominant lesions in males and

females at both 500 and 400 ppm included bilateral

symmetrical necrosis in the parietal cortex area 1 (Fig.

3C) and putamen. At 500 ppm only, bilateral symmetrical

necrosis was present in the retrosplenial cortex (Fig. 3D),

thalamus, red nucleus, posterior colliculus, nucleus of

lateral lemniscus, anterior olivary nucleus, and vestibular

nucleus. In rats exposed to 500 ppm for 12 days, there was

loss of brain substance (cavitation) within the parietal

Fig. 4. Male F344 rats exposed to 500 ppm COS for 12 days. (A) Compared to Fi

rats exposed for a longer duration. (B) The depressed area (arrows) represents an

H&E, 3.3�. (C) Similar cavitation is observed in the retrosplenial cortex (arrow), H

and immature capillary reticulum (arrow) in the area of prior malacia (cavitation)

cortex (Figs. 4A–B) and retrosplenial cortex (Figs. 4C–

D) when compared to rats exposed to 500 ppm COS for 5

days.

Twelve-week study

There were no exposure-related deaths, morbidity, or

clinical signs of toxicity in male or female rats exposed

for 12 weeks to 200, 300, or 400 ppm COS.

Body weights

Body weights of male and female rats were not signif-

icantly different from controls (P > 0.05) throughout the 12-

week study (data not shown).

g. 3B, note the prominent depression of the frontoparietal cortex (arrows) in

area of prior malacia where there is loss of the brain substance (cavitation),

&E, 10�. (D) At a higher magnification, note the cavitation, macrophages,

in the retrosplenial cortex, H&E, 50�.

Table 3

Incidence of neuropathological lesions in rats exposed to COS for 2 weeks

CNS regiona Neuropathology lesion COS concentration (ppm)

Male Female

Control 300 400 500 Control 300 400 500

Brain lesion

Parietal cortex area 1 Cortical necrosis 0/10 0/10 5/10* 6/6** 0/10 1/10b 8/10** 10/10**

Retrosplenial cortex Cortical necrosis 0/10 0/10 0/10 4/6** 0/10 0/10 0/10 7/10**

Hippocampus CA1 and 3 Neuronal necrosis 0/10 0/10 1/10 0/6 0/10 0/10 1/10 3/10

Putamen Necrosis 0/10 0/10 5/10* 6/6** 0/10 0/10 6/10** 8/9**

Thalamus Necrosis or vacuolation 0/10 0/10 0/10 2/6 0/10 0/10 0/10 6/10**

Red nucleus Necrosis 0/8 0/9 0/10 3/6 0/10 0/9 0/8 3/8

Posterior colliculus Necrosis 0/10 0/10 2/7 3/3** 0/8 0/0 3/9 8/10**

Anterior olivary nucleus Necrosis 0/10 0/10 0/10 5/6** 0/10 0/10 0/10 6/10**

Vestibular nucleus Necrosis 0/10 0/10 0/10 2/4 0/10 0/10 0/10 1/10

Fifth cranial nerve tract Vacuolation 0/10 0/10 0/10 1/6 0/10 0/10 0/10 0/10

Nucleus lateral lemniscus Necrosis 0/10 0/0 0/0 0/0 0/0 0/0 0/0 3/5

Some animals in the 500 ppm groups became moribund and were euthanized early.a Thirty-four specific named areas of brain were recorded in the data sheets of six standard sections of the brain.b Number of animals with the lesion/number of animals for which that area of brain was examined.

*P < 0.05 vs. controls (Fisher’s exact test).

Table 4

COS exposure inhibits cytochrome oxidase (CO) activity in posterior

colliculus of rat brain

COS Female Male

(ppm)CO activitya Percentage

of control

Co activitya Percentage

of control

Day 24

0 1792 F 272b 100 1919 F 192 100

200 1477 F 281* 82 1735 F 97 90

300 1389 F 217** 78 1791 F 223 90

400 1300 F 208** 72 1676 F 202* 87

Day 52

0 1633 F 337 100 1596 F 238 100

200 1253 F 271** 77 1421 F 298 89

300 998 F 102** 61 763 F 139** 48

400 1208 F 276** 74 739 F 103** 46

Day 86

0 1931 F 265 100 1788 F 94 100

200 1607 F 167** 83 1533 F 128** 86

300 1330 F 93** 69 1283 F 106** 72

400 1249 F 219** 65 1241 F 123** 69

a Amol/min/mg/protein.b Means F SD (10).

*P < 0.05 vs. control (Dunnett’s test).

**P < 0.001 vs. control (Dunnett’s test).

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145 139

Clinical pathology

Serum chemistry measurements were evaluated after

exposure of rats for 12 weeks. Statistically significant

(P < 0.05) chemical-related changes were observed in

several serum chemistry parameters in male rats only.

Serum ALP, cholesterol, SDH, protein, and creatinine

were all significantly decreased in COS-exposed male

rats relative to controls; however, the biological signifi-

cance of these mild, negative changes is not clear (data

not shown).

Brain cytochrome oxidase activity

A concentration-related decrease in cytochrome oxidase

activity was detected in the posterior colliculus (Table 4)

and parietal cortex (Table 5) of male and female rats

exposed to COS for 3, 6, and 12 weeks. These significant

decreases (P < 0.05) in cytochrome oxidase activity were

present in the brain of rats exposed to 200 and 300 ppm

where no histopathological findings were present. No gen-

der-related differences in cytochrome oxidase activity were

apparent except at day 52. Cytochrome oxidase activity was

decreased to less than 50% of controls in male rats at day

52; however, animals appeared to be recovering by the 86-

day time point.

Functional observational battery

Mild gait changes were observed in only about a fourth

of the rats, and this was somewhat more prevalent at 6

weeks compared to 12 weeks. All exposed males displayed

decreased handling reactivity, but only at 6 weeks. Increased

arousal was also observed in both sexes, but this was not

consistent with respect to dose or sex. Female rats in the low

dose group only showed a decreased response to the click

stimulus. Unlike the 2-week study, grip strength changes

and hypotonia were not observed.

**P < 0.001 vs. controls (Fisher’s exact test).

Histopathology

Microscopically at 12 weeks, neuropathological findings

were only present in male and female rats exposed to 400

ppm COS (Table 6). Predominant findings included unilat-

eral and bilateral symmetrical cortical necrosis and cavita-

tion in the parietal cortex area 1 (Figs. 5A–D), and bilateral

symmetrical neuronal loss with microgliosis and sometimes

hemorrhage in the posterior colliculus (Figs. 5E–F). Occa-

Table 5

COS exposure inhibits cytochrome oxidase (CO) activity in parietal cortex

of rat brain

COS Female Male

(ppm)CO activitya Percentage

of control

CO activitya Percentage

of control

Day 24

0 1829 F 163b 100 1841 F 170 100

200 1642 F 88** 90 1580 F 204* 86

300 1129 F 127** 62 1258 F 190** 68

400 1182 F 104** 65 1066 F 234** 58

Day 52

0 2131 F 257 100 1898 F 334 100

200 1629 F 209** 76 1755 F 139 92

300 1171 F 232** 55 1277 F 108** 67

400 1227 F 139** 58 1227 F 94** 65

Day 86

0 1711 F 125 100 1687 F 214 100

200 1268 F 232** 74 1349 F 111** 80

300 928 F 175** 54 816 F 129** 48

400 857 F 72** 50 935 F 185** 55

a Amol/min/mg/protein.b Means F SD (10).

*P < 0.05 vs. control (Dunnett’s test).

**P < 0.001 vs. control (Dunnett’s test).

Table 6

Incidence of neuropathological lesions in rats exposed to COS for 12 weeks

CNS regiona Neuropathology COS concentration (ppm)

lesionControl 300 400

Parietal

cortex area 1

Cortical necrosis

or cavitation

0/10b 0/10 5/10*

Putamen Necrosis or

cavitation

0/10 0/10 2/10

Thalamus Necrosis 0/10 0/10 1/10

Posterior

colliculus

Neuronal loss or

microgliosis

0/9 0/9 7/9**

Posterior colliculus Hemorrhage 0/9 0/9 2/9

Lat. anterior

olivary nucleus

Neuronal loss or

microgliosis

0/10 0/9 1/10

Parietal cortex area 1 Cortical necrosis 0/10c 0/10 4/10*

Putamen Necrosis 0/10 0/10 0/10

Thalamus Necrosis 0/10 0/10 0/10

Posterior colliculus Neuronal loss or

microgliosis

0/9 0/9 5/9**

Posterior colliculus Hemorrhage 0/9 0/9 1/9

Lat. anterior

olivary nucleus

Neuronal loss or

microgliosis

0/9 0/10 0/9

a Thirty-six specific-named areas of brain were recorded in the data sheets

of six standard sections of brain.b Number of male rats with the lesion/number for animals for which that

area of brain was examined.c Number of female rats with the lesion/number of animals for which that

area of brain was examined.

*P < 0.05 vs. controls (Fishers exact test).

**P < 0.001 vs. control (Fisher’s exact test).

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145140

sionally, necrosis was present in the putamen, thalamus, and

anterior olivary nucleus of male rats.

Brainstem auditory evoked responses

Exposure to 400 ppm COS for 2 weeks altered the

BAER waveforms relative to air-exposed controls (Fig. 6).

Changes in peak amplitudes were observed in the portion

of the waveforms associated with auditory transmission in

the brainstem. The most striking change was the reduction

in positive voltage associated with peak P4 (F[1,18] = 9.43,

P = 0.0066) and the reduced negative voltage associated

with peak N5 (F[1,18] = 5.43, P = 0.0316). The reduction

in positive voltage at the time of peaks N3 (F[1,18] =

16.79, P = 0.0007) and N4 (F[1,18] = 8.42, P = 0.0095)

resulted in a larger negative response in treated animals

(Fig. 7).

Changes in the latencies of BAER peaks were less

dramatic than changes in peak amplitudes. Increases in the

latencies of peaks P2 (F[1,18] = 4.56, P = 0.0467), N3

(F[1,18] = 17.08, P = 0.0006), P4 (F[1,18] = 5.40, P =

0.0320), N4 (F[1,18] = 5.27, P = 0.0339), and P5 (F[1,18] =

8.42, P = 0.0095) were observed (Fig. 7).

Exposure to 400 ppm COS produced only minor changes

in the animal’s general health status. No changes in colonic

temperature (F[1,18] = 0.90, P = 0.3559) were observed.

The colonic temperatures were 39.0 F 0.1 and 38.7 F 0.3

jC for the control and 400 ppm animals, respectively.

Magnetic resonance microscopy (MRM)

The incorporation of magnetic resonance microscopy

(MRM) in this study allowed the identification of signif-

icant lesions that would have been missed with standard

histological evaluation of three brain sections (Sills et al.,

2004). In the initial range-finding study, three standard

brain sections were evaluated from each animal, and

functional sites associated with the auditory system were

identified as potential targets of COS neurotoxicity. Be-

cause the posterior colliculus of only one animal was

examined, it was not apparent that this site was a signif-

icant target of COS toxicity. However, after conducting

parallel studies and evaluating the entire brain by MRM, it

became clear that the posterior colliculus was indeed a

primary target (Fig. 8). After evaluating the MRM data,

the fixed tissues were sectioned to include the posterior

colliculus and this site was confirmed by light microscopy

to be a consistent site of neurotoxicity.

Discussion

The brain has been reported to be the major target organ

in acute inhalation toxicity studies of COS in rats (Mon-

santo, 1985; Nutt et al., 1996). The current study examined

in greater detail the distribution of lesions in the brain

following COS exposure for up to 12 weeks using standard

histopathology and MRM (Sills et al., 2004). The target

sites observed were dependent upon the COS concentration

and duration of exposure. Acute exposures of rats to 600

Fig. 5. Female F344 rats exposed to 400 ppm COS for 12 weeks. (A) The MRM image shows a unilateral hypointense area (arrow) in parietal cortex area 1. (B)

Matching H&E section showing an area of neuronal loss (arrow), 2.5�. (C) Higher magnification (25�) of (B) showing complete loss of the grey matter with

only a gliovascular reticulum remaining (arrow) in an area of prior malacia. (D) Note here in another example the bilateral symmetrical loss of brain substance

in an area of prior malacia in the parietal cortex area 1 (arrows; 2.5�). (E) In the posterior colliculus, there is bilateral symmetrical loss of neurons and

infiltration of the affected regions by reactive glial cells (arrows), H&E; 5�. (F) High magnification of (E) (50�) showing the loss of neurons. The area of

necrosis is infiltrated with glial cells and occasional microglial cells containing hemosiderin (arrows).

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145 141

ppm COS for 1 day and held for 2 weeks caused necrosis

predominantly in functional areas associated with move-

ment such as the cerebellum and associated tracts. Consis-

tent with these findings, the exposed rats showed motor

dysfunction including ataxia and head tilt. With longer

exposure to 600 ppm (2 days), consistent neurotoxicity

was seen in parietal cortex area 1, thalamus, posterior

colliculus, anterior olivary nucleus, and cerebellar roof

nucleus. Lesions observed during 2 weeks of COS exposure

to 500 ppm, but not to 400 ppm, included necrosis in the

retrosplenial cortex, thalamus, red nucleus, anterior olivary

nucleus, and nucleus of the lateral lemniscus. After 12

weeks of exposure to 400 ppm, necrosis was identified in

parietal cortex area 1, the posterior colliculus, and to a lesser

extent in the putamen, thalamus, and anterior olivary nu-

cleus. High exposure concentrations (400 and 500 ppm)

produced motor dysfunction in rats, but following lower

concentrations (200 and 300 ppm), there was no clear

pattern of neurobehavioral changes. Compensation of these

behavioral effects was apparent at high concentrations, since

effects after 2 weeks of exposure to 400 ppm were greater

than after 6 or 12 weeks at the same concentration.

Carbonyl sulfide first caused necrosis of neurons fol-

lowed by neuronal loss with some cavitation and collapse of

the brain tissue. In affected areas, there was spongiosis of

the residual neuropil and the presence of hemosiderin-laden

macrophages. Additionally, there was a reactive astrocytic

reticulum and proliferation of capillaries in areas of prior

necrosis. For the most part, even with repeated COS

exposures, these lesions appeared to develop early and there

Fig. 6. Average BAER waveforms following stimulation with an 80 dB

SPL rarefaction click for rats exposed to 0 or 400 ppm COS (n = 10 per

group) for 2 weeks. Waveforms are plotted with positivity upward. The

shaded and crosshatched regions represent the 95% amplitude confidence

intervals for the waveforms from 0 and 400 ppm COS rats, respectively (see

text for details).

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145142

was no evidence of later sequential development of new

lesions. These results suggest that vulnerable sites were

affected early and unaffected areas of brain may be refrac-

tory to lesions by this compound.

The distribution of lesions within the brain may be

multifactorial and a reflection of function, regional metabolic

and biochemical differences, and possibly blood supply

(Cavanagh, 1988; Cremer et al., 1983). The major sites of

COS toxicity were associated with key CNS functions; the

parietal cortex area 1 is associated with somatosensory

function particularly of the vibrissae, face, and forelimbs,

the thalamus is the main sensory relay to cortical areas, the

retrosplenial cortex is associated with limbic function, and

the anterior olivary nucleus, lateral lemniscus, and posterior

colliculus are linked to function of binaural sound localiza-

tion and reflexes, respectively (Paxinos, 1995). Corres-

ponding to its functional role, the posterior colliculus is

Fig. 7. Amplitudes and latencies of BAER peaks (FSE) were affected by exposure

COS reduced the positive voltage associated with peaks N3, P4, and N4, and red

increased peak latencies (see text for details). *P < 0.05, significantly different fr

one of the most metabolically active areas in the brain and

has the largest microvascular blood volume (Cremer et al.,

1983; Scremin, 1985), the highest rate of blood flow (Sakur-

ada et al., 1978), and the highest rate of glucose metabolism

(Sokoloff, 1981; Sokoloff et al., 1977). The posterior colli-

culus may be the most metabolically active area in the brain

because auditory processing is continuous (Faingold et al.,

1991; Webster, 1995). Because of their acute hearing capac-

ity, rodents tend to show lesions in the posterior colliculus

but not in the superior colliculus due to the relative impor-

tance of auditory vs. visual sensory processing.

In this study, exposure to 400 ppm COS for 2 weeks

resulted in functional changes in auditory processing at the

level of the brainstem. Peaks of the BAER are believed to

be generated in the following region(s): peak P1 by the

auditory nerve, peak P2 at the level of the cochlear nucleus,

peak P3 in the region of the olivary complex, peak P4 in the

region of the lateral lemniscus, peak P5 in the brainstem

and posterior colliculus, and P6 in the brainstem and medial

geniculate nucleus (Chen and Chen, 1991; Melcher et al.,

1996; Moller and Jannetta, 1986; Shaw, 1988; Zaaroor and

Starr, 1991). The observed decrease in peak P4 amplitude is

consistent with a lesion in the region of the olivary

complex–lateral lemniscus region of the brainstem. Of

interest is the general decrease in positivity over the N3–

P5 region and the reduction in negativity over the N5–P6region of the BAER waveforms (Fig. 6). Reducing the high

frequency content of BAER waveforms using filters yields

a slow positive (SP3) and slow negative (SN5) waveform

during these portions of the BAER (Shaw, 1987). The SP3

wave has been proposed to arise from activity in the lateral

lemniscus or the posterior colliculus (Hashimoto et al.,

1981; Moller and Jannetta, 1982) while the SN5 is believed

to be generated at the level of the posterior colliculus

(Hashimoto, 1982; Hashimoto et al., 1981; Funai and

to 400 ppm COS for 2 weeks. Peaks are labeled as in Fig. 6. Treatment with

uced the negative voltage associated with peak N5. Exposure to COS also

om 0 ppm control.

Fig. 8. Female F344 rat exposed to 400 ppm COS for 8 weeks. (A) MRM of whole brain (3D Image) showing the area of the brain that corresponds to the

posterior colliculus (red line). Also, note the hyperintense areas in the parietal cortex area 1 representing areas of malacia (arrows). (B) Sagittal slice through the

posterior colliculus showing a hypointense locally extensive lesion (arrow). (C) The corresponding transverse slice shows the hypointense area (arrow). (D)

Correlation of the MRM image with an H&E section confirms that the unilateral hypointense area represented a prior area of hemorrhage. The posterior

colliculus nucleus is replaced by numerous hemosiderin laden macrophages (black focal area—arrow), H&E, 2.5�.

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145 143

Funasaka, 1983; Moller and Jannetta, 1982). While these

waves were not specifically quantified in this study, the

observed changes in the BAER waveforms are consistent

with alterations in these regions of the brainstem. There-

fore, the alterations in BAERs indicate that treatment with

COS produced changes in brainstem processing of the click

stimulus from the region of the olivary nucleus to the

posterior colliculus.

The increases in BAER peak latencies were less dramatic

than the alterations in peak amplitudes. Treatment with COS

did not alter colonic temperature. Therefore, the increases in

peak latencies were not attributable to temperature-related

effects. Changes in peak latency may be the result of

alterations in waveform shape, a change in the size distri-

bution of the axons contributing to the evoked response, or

damage to the myelin (Mattsson et al., 1992). In view of the

neuronal damage in the brainstem produced by treatment

with COS, several of the above factors could contribute to

the observed increases in BAER peak latencies. Because the

affected peaks were related to brainstem generators, a

general effect by COS on myelin is unlikely (e.g., peak P1[auditory nerve] was not affected). A more complete exam-

ination of auditory electrophysiology following treatment

with COS is forthcoming and will allow replication of the

changes reported in this study.

The mechanism(s) for COS neurotoxicity is unknown.

However, in initial acute COS exposures (Nutt et al.,

1996), elevated oxyhemoglobin levels were observed in

COS exposed rats, and it was hypothesized that COS

inhibited mitochondrial respiration. Others (Chengelis and

Neal, 1980) proposed that COS is initially metabolized by

carbonic anhydrase to hydrogen sulfide (H2S), and in

subsequent studies H2S was shown to inhibit brain cyto-

chrome oxidase (Dorman et al., 2002; Khan et al., 1990;

Nicholls and Kim, 1982). Cytochrome oxidase is a rate-

limiting enzyme in the mitochondrial respiratory chain

(oxidative phosphorylation) (Nicklas et al., 1992). In

COS-exposed rats, decreases in mitochondrial cytochrome

oxidase activity were detected in the frontoparietal cortex

and posterior colliculus. Although these results suggest

that inhibition of oxidative phosphorylation may contribute

to neuronal cell death, further research is needed. These

cumulative data suggest that COS inhalation may cause

neurotoxicity by a mechanism involving inhibition of brain

cytochrome oxidase, resulting in decreased ATP production

and neuronal cell death. The ability of COS to react

D.L. Morgan et al. / Toxicology and Applied Pharmacology 200 (2004) 131–145144

directly with critical target sites in the brain is not known;

however, H2S and the hydrosulfide anion (HS�) are

potential metabolites of COS (Chengelis and Neal, 1980)

and are potent inhibitors of cytochrome oxidase (Holland

and Kozlowski, 1986; Smith et al., 1977). Additional

research is needed to investigate the potential role of

H2S in COS neurotoxicity.

The distribution of brain lesions caused by COS is

similar to that described for other chemicals known to

target the mitochondrial respiratory pathway (Cavanagh,

1988, 1993). These vulnerable areas of the brain all have

relatively high resting glucose utilization rates (Bagley et

al., 1989; Sokoloff, 1981; Sokoloff et al., 1977). Exposure

to 1,3-dinitrobenzene (DNB), a commonly used industrial

chemical, caused lesions similar to those of COS in the

anterior olivary nuclei, posterior colliculus, and cerebellar

nucleus by a mechanism that inhibits oxidative phosphor-

ylation (Philbert et al., 1987; Ray et al., 1992). Chlorohy-

drin also caused similar lesions at sites in the brain that

have high-energy requirements (Cavanagh and Nolan,

1993; Cavanagh et al., 1993).

In conclusion, inhalation exposure to COS at concen-

trations of 400 ppm and above resulted in marked neuro-

toxicity. Carbonyl sulfide targeted specific neuroanatomical

sites in the auditory system that correlated with alterations

in the amplitude of BAER between the anterior olivary

nucleus to the medial geniculate nucleus. The higher the

exposure concentration, the more striking and widespread

were the neuropathological effects as well as the neuro-

behavioral effects. Our data suggest that decreases in

cytochrome oxidase in exposed rats may be involved in

the pathogenesis of neuronal injury. Neuropathological

findings were not present at lower COS concentrations

(200 and 300 ppm), and no clear behavioral changes were

observed. Cytochrome oxidase activity was decreased at the

lower concentrations; however, not to a point where cell

death occurred.

Acknowledgments

The ManTech Environmental Technology, Inc. personnel

are acknowledged for their expertise in conducting the

inhalation exposures. Drs. Fletcher Hahn and Janet Benson

are thanked for useful discussions and for providing data

from the initial COS studies conducted at Lovelace

Respiratory Research Institute. Dr. Connie Cummings of

Pathology Associates, Division of Charles Rivers is

recognized for her superior coordination of the cardiac

perfusion team. The authors also wish to thank Drs. Irwin

and Boyes for their crucial review of the manuscript, Ms.

Jaimie Graff and Pamela Phillips for excellent technical

support in the BAER testing and neurobehavioral testing,

respectively, and Mr. C. Hamm for the design and

implementation of the software used for electrophysiolog-

ical recordings.

The information in this document has been funded in part

by the U.S. Environmental Protection Agency. It has been

subjected to review by the National Health and Environmen-

tal Effects Research Laboratory and approved for publication.

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