Short-term oral toxicity of butyl ether, ethyl hexyl ether,methyl heptyl ether and 1,6-dimethoxyhexane
in male rats and the role of 2-methoxyacetic acid
Raymond Poona,∗, Michael Wadea, Victor E Valli b, Ih Chua
a Environmental Health Science Bureau, Health Canada, Ottawa, Ont., Canada K1A 0L2b College of Veterinary Medicine, University of Illinois, Urbana, USA
Received 17 February 2005; received in revised form 6 June 2005; accepted 13 June 2005Available online 2 August 2005
Abstract
A 4-week oral study was conducted in male rats to characterize and compare the toxicity of four aliphatic ethers (butyl ether,BE; ethyl hexyl ether, EHxE; methyl heptyl ether, MHpE; and 1,6-dimethoxyhexane, DMH) which have been proposed ashigh-cetane diesel additives. Male Sprague-Dawley rats (280± 20 g) were divided into groups of seven animals each and wereadministered by gavage low (2 mg/kg body weight), medium (20 mg/kg) or high (200 mg/kg) doses of BE, EHxE, or MHpE, 5days per week for 4 weeks. Another group of animals was administered DMH at 200 mg/kg while the control group receivedthe vehicle (corn oil at 1 ml/100 g bw) only. At the end of the treatment period, relative testis weights and thymus weights were
vealed. Urinarybut not inis, while0 mg/kgroninecreased
esorufin-wnut not inared toncludedt high
significantly decreased in the DMH group but not in animals receiving BE, EHxE, or MHpE. Microscopic examination redegeneration of the seminiferous tubules and reduction of sperm density in the epididymides in the DMH treatment groupcreatine/creatinine ratio, a sensitive indicator of testicular damage, was markedly elevated in the DMH treated animalsthose treated with BE, EHxE, or MHpE. In the bone marrow, DMH caused mild dyserythropoiesis and dysthrombopoiesBE, EHxE, and MHpE produced mild increases in granulocytes and myelocyte/erythrocyte ratio. All four ethers at 20caused mild histological changes in the thyroid but no significant modulation in the circulating thyroxin (T4) or triiodothy(T3) levels. All four ethers produced hepatic effects at 200 mg/kg consisting of mild, adaptive histological changes, inurinary ascorbic acid output, and elevation in the activities of one or more xenobiotic metabolizing enzymes (benzyloxyrO-dealkylase, UDP-glucuronosyltransferase, glutathione-S-transferases). The level of 2-methoxyacetic acid (MAA), a knotesticular and developmental toxin, was significantly increased in the urine and plasma of animals treated with DMH bthose administered the high dose BE, EHxE, or MHpE. Amomg the individual rats treated with DMH, the MAA level appecorrelate with the severity of toxicity such as testicular and thymic weights, and urinary creatine/creatinine ratio. It is cothat BE, EHxE, and MHpE differed from DMH in that they did not produce testicular or thymic toxicity. All four ethers a
Because of their chemical characteristics and misci-bility properties, liquid alkyl ethers are potentially idealcetane1 boosters. Their presence in diesel improvescombustion quality such as shorter ignition delayand cleaner burning. In addition, ether additives mayreduce emission of regulated combustion emissionproducts such as carbon monoxide, particulate mat-ters, and total hydrocarbons (Zhu et al., 2003; Shenet al., 1993; Heinz, 1993). Surprisingly, very littletoxicological research has been done on this type ofaliphatic ethers even though presence or absence oftoxicity should be a foremost consideration at theproduct development stage. The acute oral effectsof dimethoxymethane (methylal), one of the earliestcandidate diesel additives, have been investigated infemale rats. The study revealed treatment effects suchas transient ataxia and raised a concern of the pos-sible hydrolysis of dimethoxymethane to formalde-hyde and methanol at acidic conditions in the stomach(Poon et al., 2000). This study was followed by amore comprehensive toxicity study on three candidate
and an occupational health concern. Because contactwith higher levels of diesel additives is more commonin work environments, a 5 days per week for 4 weeksdosing regimen was adopted to reflect such an exposurescenario. DMH, a testicular toxin (Poon et al., 2004),was included in the study for comparison (seeFig. 1for structure and synonyms of test compounds andstructurally related testicular toxins). An expanded testprotocol was adopted that included the measurementof urinary creatine as a sensitive biomarker of testicu-lar injury (Nicholson et al., 1986; Moore et al., 1992),and thyroxin and triiodothyronine as biochemical indi-cators of thyroid effects. The second objective of thestudy is to test the hypothesis that 2-methoxyacetic acid(MAA) is the proximal toxicant responsible for the tox-icity of DMH. In the previous study it was noted that thetoxic expressions of DMH, such as testicular atrophy,degeneration of seminiferous tubules, and thymic atro-phy were similar to that elicited by 2-methoxyaceticacid (Miller et al., 1982, 1984; Moss et al., 1985). Ithas been shown that glycol ethers such as ethylene gly-col monomethyl ether (EGMME) elicited multitudesof toxic effects through the metabolite MAA (Foster
lethy-eiro-87
ethers: pentyl ether (PE), 1,4-diethoxybutane (DEB),and 1,6-dimethoxyhexane (DMH). DMH was identi-fied as a testicular toxin producing testicular atrophyand epididymal hypospermia (Poon et al., 2004). It alsocaused thymic atrophy, liver and thyroid changes and
et al., 1984; Miller et al., 1984). More complex glycoethers such as ethylene glycol dimethyl ether andlene glycol dimethyl ether (EGDME) also exerted thtoxicity through MAA as the proximal toxic metablite (Daniel et al., 1991; Hardin and Eisenmann, 19).
thrombocytopenia.The objectives of the present study were two-fold.
F es:b hylh rizet ent.P blic
dro-c ualityo alityo etanei e fuelb
In the present study, MAA was measured in both urineand plasma of animals treated with BE, EHxE, MHpEa thet
2
2
er( ni-v ane
irstly, toxicity study of three candidate ether additivutyl ether (BE), methyl heptyl ether (MHpE) and etexyl ether (EHxE), were undertaken to characte
heir toxicity and to provide data for risk assessmotential exposure to diesel additives is both a pu
1 Cetane, orn-hexadecane (C16H34) is a colorless, liquid hyarbon and a reference fuel used to determine the ignition qf diesel fuels. Cetane number is a measure of the ignition quf a diesel fuel that represents the percentage by volume of c
n �-methylnapthalene that gives the same ignition delay as theing tested.
nd DMH. The MAA levels were then related tooxicological observations.
. Materials and methods
.1. Materials
Ethyl hexyl ether (EHxE) and methyl heptyl ethMHpE) were synthesized by Dr. Kaliaguine, Uersit́e Laval, Quebec, Canada. 1,6-Dimethoxyhex
R. Poon et al. / Toxicology 214 (2005) 99–112 101
Fig. 1. Structure and synonyms of test compounds and known testicular toxins related to glycol ethers.
(DMH) was synthesized by Wychem Ltd. (Suffolk,England). Butyl ether (BE) and 2-methoxyacetic acid(MAA) were purchased from Aldrich Chem Co. (Wis-consin, USA). The purity of the above ethers was >99%by gas chromatography.
2.2. Animal treatment
Seven-week-old (280± 20 g) male Sprague-Daw-ley rats (Charles River Laboratories, St. Constant, Que-bec, Canada) were divided into 11 groups of 7 animalseach. They were housed in individual Health Guard Rcages (Research Equipment Co., Bryant, Texas) andwere given food and water ad libitum. After 1-weekacclimatization, the groups were administered, by gav-age, the test susbstances (BE, EHxE, MHpE) in cornoil in one of the following doses: 2 mg/kg bw (low),20 mg/kg bw (medium), and 200 mg/kg bw (high). Onegroup of animals was administered DMH at 200 mg/kgbw and control animals were administered corn oil(1.0 ml/100 g bw) only. The doses were administeredas a single dose, 5 days per week, for four consecu-tive weeks. Body weights and food consumption were
measured weekly. Cage-side observations were madeweekly. The battery of observations included: pos-ture, clonic movement, gait score, piloerection, pito-sis, lacrimation, salivation, vocalization, and ease ofremoval from home cage.
Overnight urine was collected from each animalscaged individually at the end of 4th week. A portionof the urine was stored at−80◦C for the determina-tion of MAA by the method described below, proteinby the Lowry procedure, andN-acetylglucosaminidase(NAGA) activity (Poon et al., 1995). The remainingurine was preserved in 0.4 N HCl/10 mM EDTA (finalconcentrations) for the determination of ascorbic acid.
At termination, the animals were anesthetized withinhaled Isoflurane®. Blood was withdrawn from theabdominal aorta for hematological analysis, and prepa-ration of plasma for 2-methoxyacetic acid analysisand serum for clinical chemistry. The right lung wasclamped and the left lung was instilled with saline(17.5 ml/kg bw) according to the method ofHatch et al.(1986). The lavage fluid was centrifuged at 700×g for15 min and the cell-free bronchoalveolar lavage fluid(BALF) was stored at−70◦C pending the analysis
102 R. Poon et al. / Toxicology 214 (2005) 99–112
for protein and NAGA activity. The brain, heart, thy-mus, liver, kidneys, spleen, and testis were excised andweighed. A 2 g piece of liver was removed and homog-enized in 3 ml of 0.05 M Tris/0.15% KCl buffer, pH 7.4.Part of the homogenate was centrifuged at 9000×g for20 min to obtain the S9 fraction. Both the homogenatesand S9 fractions were stored at−80◦C until use.
2.3. Histopathology
The liver, thymus, thyroid, bone marrow from rightfemur and epididymis were preserved in phosphate-buffered formalin The right testis was fixed in Bouin’ssolution, washed in 70% ethanol and preserved inphosphate-buffered formalin. The fixed tissues anddecalcified bone marrow were dehydrated with gradedalcohol, cleared and impregnated in paraffin. The paraf-fin blocks were sectioned to 5�m thickness and stainedwith hematoxylin and eosin for microscopic examina-tion.
2.4. Hematological and biochemical analysis
A Technicon H1E hematology analyzer (Bayer,Toronto, Canada) was used for the determination ofthe following hematological parameters: erythrocytecount, hematocrit, mean corpuscular volume, meancorpuscular hemoglobin, platelet count, white bloodcell counts and percent lymphocyte. A Technicon RA-XT analyzer was used to obtain the following serumc artatea tein,a eati-n T4)a g ar da).
liverh al-b ives ands thod( edb bySu red m.Ce as
measured by an HPLC procedure (Poon et al., 1994).Urinary creatine was measured using a modified Rochecreatinine PAP assay kit (Roche Diagnostics, Quebec,Canada) in which the creatinase was omitted in thecomplete assay mixture so that the final absorbancerepresented creatine only (Gray et al., 1990). All uri-nary analytes concentrations were normalized againsturinary creatinine as measured by the PAP creatinineassay.
The liver 9000×g supernatant was assayed forthe activity of ethoxyresorufin-O-deethylase (EROD)(Lubet et al., 1985), pentoxyresorufin-O-dealkylase(PROD), benzyloxyresorufin-O-dealkylase (BROD)(Burke et al., 1985) and glutathione-S-transferases(GST) (Habig et al., 1974). Liver homogenate wasassayed for the activity of UDP-glucuronosyltransfe-rase (UDPGT) (Burchell and Weatherill, 1981).
2.5. Analysis of 2-methoxyacetic acid
2-Methoxyacetic acid (MAA) in urine and plasmawas determined using a dual column HPLC methodas described below. A 0.5 ml aliquot of urine wasmixed with 0.5 ml of 50 mM Tris–HCl, pH 8.1, andloaded onto a Bond Elut SAX anion exchange col-umn (500 mg, Varian Analytical Instruments, Missis-sauga, Canada). The SAX column was washed with1.0 ml 25 mM Tris–HCl, pH 8.1 and then eluted withexactly 1.0 ml 0.5 N HCl. For plasma samples, 0.25 mlplasma was mixed with an equal volume of 50 mMT lo-r l-lu wasl col-u entym ntoa VPd tion-a rsilO ,U exHR -v ratew eaka inp
hemistry parameters: lactic dehydrogenase, aspminotransferase, alkaline phosphatase, total prolbumin, inorganic phosphate, urea nitrogen, crine, glucose and cholesterol. Serum thyroxin (nd triiodothyronine (T3) were determined usinadioimmunoassay kit (Medicorp, Montreal, Cana
Reduced glutathione (GSH) was measured inomogenates using a colorimetric kit method (Ciochem, La Jolla, CA). Thiobarbituric acid-reactubstances (TBARS) in the liver homogenateserum were determined by a fluorescence meYagi, 1982). Protein carbonyl content was determiny a 2,4-dinitrophenylhydrazine method describedtarke et al. (1987). Protein and NAGA activity inrine and cell-free bronchoalveolar fluid (BALF) weetermined with a Micro Lowry method (Sigma Cheo. Missouri, USA) and a colorimetric method (Poont al., 1995), respectively. Urinary ascorbic acid w
ris–HCl, pH 8.1 and then vortexed with 1.0 ml choform. The mixture was kept in ice for 15 min foowed by centrifugation at 400×g for 10 min. Thepper layer representing the deproteinized plasma
oaded onto a Bond Elut SAX anion exchangemn and eluted as described for the urine. Twicrolitres of the 0.5 N HCl eluate was injected iShimadzu HPLC system equipped with a Class
ata acquisition system. The specimens were fracted in two columns arranged in series: a HypeDS column, 5�, 250× 4.6 mm (Alltech, IllinoisSA) at room temperature followed by an AminPX-87 H ion exchange column, 300× 7.8 mm (Bio-ad, Mississauga, Canada) at 40◦C. The elution solent was 0.01% phosphoric acid and the elutionas 0.5 ml/min. MAA was eluted as a discrete pt around 31 min. The detection limit is 0.05 mMlasma.
R. Poon et al. / Toxicology 214 (2005) 99–112 103
2.6. Statistical analysis
One way analysis of variance (Dunnett’s method) oranalysis of variance on rank (Student-Newman-Kuelsmethod) were used to identify groups that were signif-icantly different from the control at thep< 0.05 level.
3. Results
3.1. Gross changes and organ weights
All animals survived the 4-week treatments withno abnormal cage-side observations. Rats administered200 mg/kg bw of DMH consumed 20% less feed thanthe control (109± 8 g/week versus 136± 13 g/week;p< 0.05) over the four weeks treatment period. Themean final body weight of the DMH group was17% lower than that of the control (366± 22 versus429± 22, p< 0.05). The food intake and final bodyweight of all other treatment groups were not signif-icantly different from that of the control.
Relative thymus weight and testis weight of theDMH group was 48 and 21% lower than the control(p< 0.05), respectively (Table 1). When the relativetestis and thymus weights of individual animals inthe DMH treatment groups were compared, a signif-icant positive correlation was found (thymus weight =0.00169 + 5.865× testes;R2 = 0.799,p= 0.007). Rel-ative thymus weight was also significantly depressedi xEg ted ino
3
l inat redc atan MHg acha ro-g tlyd activ-i iumd t-r evels Ta
ble
1R
elat
ive
orga
nw
eigh
ts(%
bw)
inm
ale
rats
follo
win
g4-
wee
ktr
eatm
entw
ithal
kyle
ther
s(m
ean
±S
.D.o
fsev
enan
imal
spe
rgr
oup)
Dos
e(m
g/kg
)Li
ver
Kid
neys
Hea
rtS
plee
nA
dren
als
aT
hym
usVe
ntra
lpro
stat
eaE
pidi
dym
isTe
stis
Con
trol
03.
72±
0.21
0.69
±0.
030.
28±
0.02
0.17
±0.
020.
012±
0.00
20.
142±
0.01
70.
311±
0.05
90.
275±
0.04
00.
770±
0.06
9
BE
23.
56±
0.20
0.68
±0.
050.
30±
0.02
0.16
±0.
01nd
0.12
2±0.
017
0.28
5±0.
049
0.26
8±0.
033
0.76
3±0.
078
203.
55±
0.09
0.68
±0.
030.
28±
0.02
0.16
±0.
02nd
0.14
1±0.
023
0.24
1±0.
049
0.26
8±0.
015
0.75
6±0.
047
200
3.84
±0.
300.
69±
0.03
0.29
±0.
020.
17±
0.02
0.01
1±0.
002
0.11
8±0.
009
0.27
3±0.
038
0.24
9±0.
020
0.69
3±0.
067
EH
xE2
3.58
±0.
220.
69±
0.06
0.30
±0.
030.
17±
0.03
nd0.
124±
0.02
40.
272±
0.06
80.
281±
0.02
40.
816±
0.05
520
3.59
±0.
210.
68±
0.05
0.28
±0.
010.
16±
0.01
nd0.
110±
0.01
3*0.
282±
0.06
60.
261±
0.00
70.
753±
0.05
220
03.
50±
0.19
0.68
±0.
040.
29±
0.02
0.16
±0.
030.
013±
0.00
20.
133±
0.01
00.
259±
0.05
50.
286±
0.02
50.
787±
0.08
5
MH
pE2
3.56
±0.
260.
69±
0.03
0.29
±0.
010.
15±
0.02
nd0.
129±
0.01
40.
251±
0.05
20.
260±
0.02
90.
748±
0.06
920
3.55
±0.
150.
70±
0.03
0.30
±0.
020.
15±
0.01
nd0.
128±
0.02
70.
248±
0.04
00.
279±
0.02
00.
793±
0.04
120
03.
84±
0.21
0.68
±0.
030.
29±
0.01
0.17
±0.
010.
012±
0.00
20.
115±
0.01
3*0.
293±
0.05
10.
274±
0.02
70.
796±
0.05
0
DM
H20
03.
53±
0.20
0.69
±0.
030.
30±
0.03
0.16
±0.
010.
010±
0.00
20.
074±
0.01
7*0.
256±
0.03
50.
244±
0.02
40.
605±
0.11
1*
aW
eigh
toff
orm
alin
-pre
serv
edtis
sues
;nd:
notd
one.
*O
new
ayan
alys
isof
varia
nce,
sign
ifica
ntly
diffe
rent
from
cont
rola
tp
<0.
05.
n the high dose MHpE and the medium dose EHroups. No treatment-related changes were detecther tissues measured (Table 1).
.2. Hematology and serum biochemistry
Except for a slight increase in hemoglobin levenimals receiving the medium dose MHpE (Table 2),
here were no treatment-related changes in theells, white cells and differential white cell counts (dot shown). Platelet level was the lowest in the Droup, but the difference with the control did not restatistically significant level. Serum lactic dehyd
enase activity in the DMH group was significanepressed by 60%. Serum alkaline phosphatase
ty was reduced by 40% in animals treated with medose of MHpE (Table 2). No significant treatmenelated changes were observed in the serum l
104 R. Poon et al. / Toxicology 214 (2005) 99–112
Tabl
e2
Hem
atol
ogic
alan
dse
rum
bioc
hem
ical
chan
ges
inra
tsfo
llow
ing
4-w
eek
trea
tmen
twith
alky
leth
ers
(mea
n±
S.D
.ofs
even
anim
als
per
grou
p)
Dos
e(m
g/kg
)H
emat
olog
yS
erum
bioc
hem
istr
y
Hem
oglo
bin
(g/d
l)P
late
lets
(109
/L)
Asp
arta
team
inot
rans
fera
se(A
ST
)(U
/L)
Lact
ate
dehy
drog
enas
e(L
DH
)(U
/L)
Alk
alin
eph
osph
atas
e(A
LP)
(U/L
)To
talt
hyro
xin
(T4)
(�g/
dL)
Tota
ltrii
odot
hyro
nine
(T3)
(ng/
L)
Con
trol
013
.7±
0.5
975±
118
39.7±
11.4
590±
281
361±
914.
17±
0.58
61.2±
9.8
BE
214
.0±
0.4
1020
±11
738
.9±
5.7
764±
203
357±
106
3.86
±0.
5555
.4±
12.0
2014
.0±
0.5
1057
±11
435
.9±
7.9
546±
223
291±
104
3.90
±1.
0164
.3±
11.0
200
13.5±
0.6
1035
±87
36.0
±6.
941
6±11
427
7±88
4.45
±0.
7063
.1±
18.9
EH
xE2
14.0
±0.
695
2±
146
42.3±
10.1
346±
171
292±
123
3.97
±0.
5458
.1±
7.9
2013
.7±
0.6
993±
6240
.6±
9.2
516±
642
258±
953.
99±
0.91
51.5±
10.0
200
14.1±
0.6
1111
±86
34.7
±4.
147
6±24
035
2±14
33.
91±
0.84
64.4±
12.7
MH
pE2
14.2
±0.
510
24±
124
34.1±
7.8
737±
338
233±
554.
13±
0.66
56.7±
16.1
2014
.5±
0.3*
1063
±11
639
.0±
9.2
584±
282
215±
47*
3.60
±0.
7760
.3±
11.2
200
14.3±
0.3
968±
168
39.0±
4.2
670±
343
226±
724.
19±
0.58
63.8±
15.4
DM
H20
013
.7±
0.4
915±
185
32.0±
10.4
234±
143**
223±
993.
12±
0.62
58.1±
5.8
*O
new
ayan
alys
isof
varia
nce,
sign
ifica
ntly
diffe
rent
from
cont
rola
tp
<0.
05.
**A
naly
sis
ofva
rianc
eon
rank
s,si
gnifi
cant
lydi
ffere
ntfr
omco
ntro
lat
p<
0.05
.
of aspartate aminotransferase, thyroxin (T4) and tri-iodothyronine (T3).
3.3. Hepatic enzyme activity
In the liver, benzyloxyresorufin-O-deethylase(BROD) activity was significantly elevated in animalstreated with high dose BE, and GST activities wereelevated in animals treated with high dose EHxEand MHpE (Table 3). The activities of all three liverenzymes were significantly increased in DMH-treatedanimals. There were no significant treatment-relatedchanges in the EROD and PROD activities.
3.4. Urinary analytes
Urinary creatinine levels were not significantlyaffected by treatment, but urinary creatine was sig-nificantly increased only in the DMH group. As aresult, there was a 11-fold increase in the molar ratioof creatine/creatinine in the urine of the DMH treatedanimals, but not in those treated with BE, EHxE andMHpE (Fig. 2). Marked increase in urinary ascorbicacid was observed in all groups receiving the 200 mg/kgdose of BE, EHxE, MHpE and DMH, with the high-est increase of 18-fold observed in the EHxE group(Fig. 3). Protein levels andN-acetylglucosaminidaseactivities in the urine were not affected by treatment(data not shown).
F fol-l malsp
ig. 2. Molar ratio of creatine/creatinine in the urine of animalsowing 4-week treatment with alkyl ethers (mean of seven anier group± S.D.). (* ) Significant difference from control atp≤ 0.05.
R. Poon et al. / Toxicology 214 (2005) 99–112 105
Table 3Hepatic enzyme activities in rats following 4-week treatment with alkyl ethers (mean± S.D. of seven animals per group)
BROD (nmol/min/mg prot) UDP-GT (nmol/min/mg prot) GST (�mol/min/mg prot)
* One way analysis of variance, significantly different from control atp< 0.05.
3.5. Bronchoalveolar fluid analytes
No treatment-related changes were detected in theprotein levels andN-acetyl glucosaminidase activityof the cell-free broncholaveolar lavage fluids (data notshown).
3.6. Biomarkers of oxidative stress
Dose-related decreases in TBARS were observed inthe livers of all treatment groups. The most pronounceddecrease was found in the group treated with 200 mg/kg
Fig. 3. Ascorbic acid levels in the urine of animals following 4-weektreatment with alkyl ethers (mean of seven animals per group± S.D.).(*
DMH in whom TBARS was only 45% that of the con-trol (Table 4). Changes in serum TBARS generallymatched that of the liver, with significant decreasesobserved in the DMH and the high dose MHpE groups.The levels of reduced GSH in the liver were unaf-fected by treatment with BE, EHxE and MHpE. Thereappeared to be an increase in GSH in the DMH groupalthough it did not reach a statistically significant level.Protein carbonyls in the liver and serum were unaf-fected by treatments.
3.7. MAA levels and correlation with tissueweights and urinary creatine/creatinine ratio
Urinary MAA levels in the DMH treatment groupwere 6.4 times higher than that of the control but nosignificant changes were found in urine of animalstreated with BE, EHxE and MHpE (Table 5). MAAwas not detected in the plasma of controls and animalstreated with 200 mg/kg bw BE, EHxE or MHpE, butwas readily detected in animals receiving 200 mg/kgDMH with a mean plasma concentration of 0.34 mM(Table 5). When the plasma MAA of individual ani-mals in the DMH treatment group was compared withtheir respective relative testis and thymus weights, neg-ative regression lines of almost identical slopes wereobserved although the correlation did not reach statis-tical significance (Fig. 4). On the other hand, a positiveregression line was found between plasma MAA andu
) Significant difference from control atp≤ 0.05. rinary creatine/creatinine ratio (Fig. 4).
106 R. Poon et al. / Toxicology 214 (2005) 99–112
Table 4Biomarkers of oxidative stress in rats following 4-week treatment with alkyl ethers (mean± S.D. of seven animals per group)
* One way analysis of variance, significantly different from control atP< 0.05.
3.8. Histopathlogical changes
The most prominent changes were found in the tes-ticles of animals treated with DMH and consisted ofa moderate degree of degeneration of the seminifer-ous tubules that was not found in any other treatmentgroup (Table 6). The changes consisted of multifocaldegeneration of the seminiferous tubules, with sometubules possessing only one layer of cells, and inter-
Table 5Urinary and serum 2-methoxyacetic acid (MAA) following 4-weektreatment with alkyl ethers (mean± S.D. of seven animals per group)
mg/kg bw Urinay MAA(mmol/g creatinine)
Serum MAA(mM)
Control 0.58± 0.42 nd
BE0.69± 0.70 –0.63± 0.54 –0.72± 0.40 nd
EHxE0.60± 0.45 –0.52± 0.57 –0.63± 0.33 nd
MHpE0.32± 0.19 –0.46± 0.27 –0.64± 0.36 nd
DMH 3.74± 1.74* 0.34± 0.21
* One way analysis of variance, significantly different from controlatp< 0.05; nd: not detected at a detection limit of 0.05 mM; (–) notanalyzed.
stitial vacuolation (Fig. 5). Examination of the epip-didymis revealed no treatment related changes in thecaput (head) and corpus (body) sections, but a moderateto severe reduction of sperm in the cauda (tail) sec-tion in animals receiving DMH (Table 6). The severehypospermia restricted to the epididymis cauda wasaccompanied by the presence of spermatid giant cellsand cell debris. Treatment-related changes observed inthe thyroid were reduced follicle size, increased epithe-lial thickness, nuclear vesiculation and cytoplasmicvacuolation. These changes were mild to moderate inseverity and were more prominent in the medium doseMHpE group and the DMH group. Thyroid changesunique to the DMH treated animals included a mild
Table 6Histopathological changes in the liver, thyroid, kidneys, bone marrow testicles, and epididymis following 4-week oral administration of alkyl ethers
* Denotes the number of animals with histologial changes out of seven per group.** Denotes average severity index, where (1) minimal, (2) mild, (3) moderate and (4) marked. The scores were obtained by dividing the sum of total scores by the number of animals
examined. For tissue changes that are focal, locally extensive and multifocal, a score of less than integer is assigned as follows: minimal focal = 0.25, minmal locally extensive = 0.5,minimal multifocal = 0.75, mild focal = 1.25, mild, locally extensivel = 1.50, mild multifocal = 1.75.
108 R. Poon et al. / Toxicology 214 (2005) 99–112
Fig. 5. (A) Testis from a rat in the control group. The seminiferous tubules are of uniform diameter and depth of lining cells with spermatogenesisoccurring at various stages in each. (B) Testis from a rat in the high dose MHpE group. The capsule and the tubules are normal with spermat varying stages of maturation. (C) Testis from a rat treated with DMH. Tubules and lining cells vary widely in diameter and depth. There ismultifocal complete loss of spermatogonia and all progeny with only Sertoli cells remaining. Hematoxylin and eosin stain× 130.
degree of papillary proliferation and reduced colloiddensity (Table 6). Bone marrow changes observedincluded increased granulocytes and increased mye-oloid/erythroid ratio in animals treated with the BE,EHxE and MHpE, and dyserythopoiesis and dysthrom-bopoiesis in the DMH group. All bone marrow changeswere minimal in severity (Table 6). Thymus changeswere architectural in nature and were confined to ani-mals receiving DMH. Mild and reversible nuclear andcytoplasmic changes were observed in all treatmentgroups but were more prominent in the medium andhigh dose MHpE groups and the DMH group.
4. Discussion
The changes observed in the animals administered200 mg/kg DMH were identical to the adverse effectsinitially reported (Poon et al., 2004). The prominentchanges were decreased testis and thymus weights,degeneration of the seminiferous tubules, and reducedsperm density in the epididymides, and the more sub-tle changes included decreased serum LDH activityand increased hepatic GST activity. In contrast, ani-mals treated with BE, EHxE, or MHpE did not showsignificant testicular, epididymal or thymic effects.
R. Poon et al. / Toxicology 214 (2005) 99–112 109
Urinary creatine/creatinine ratio is a sensitivebiomarker of testicular damage (Nicholson et al.,1986;Gray et al., 1990; Draper and Timbrell, 1996).For example, increased urinary creatine/creatinine canbe detected in rats as early as 24-h after receiv-ing a single 500 mg/kg oral dose of ethylene glycolmonomethyl ether (Rawcliffe et al., 1989). The samecompound administered to rats via drinking water at87 mg/kg for 10 days caused an increase in urinarycreatine/creatinine at day-2 (Butterworth et al., 1995).A single dose (400–900 mg/kg) of methoxyacetic acidwas also shown to produced a significant increase inurinary creatine/creatinine in male mice as early as 24 hafter treatment (Moore et al., 1992; Traina et al., 1997).In these studies, the urinary change generally precededother testicular changes such as decreased testicularweight, decreased sperm head count, and spermatocytenecrosis. In the present 28-day study, marked increasein creatine/creatinine ratio was observed in the animalstreated with DMH at 200 mg/kg/day, but not in thegroups treated with BE, EHxE, or MHpE, lending fur-ther support to the conclusion that the test compoundshad no significant effect on the testes.
Another difference between BE, EHxE and MHpEand the toxic ether DMH is the effect on bone marrow.DMH caused mild dyserythopoiesis and dysthrom-bopoiesis while BE, EHxE and EHpE caused mildincreases in granulocytes and myelocyte/erythrocyteratio. In a previous study (Poon et al., 2004) a decreasein platelet level was detected which was consistentw sents unta sig-n
ald d top onlyt sitya lic-u reeo cantm o-n andr
ticr l.,1 ug-g hich
is associated with the glucuronide pathway of detoxi-fication, was stimulated (Linster and Van Schaftingen,2003). The hepatic response was further indicated byincreases in some hepatic xenobiotic enzyme activities,such as BROD in high dose BE and DMH, UDPGT inhigh dose DMH, and GST in high dose EHxE MHpEand DMH. Histologically, high dose of BE, EHxE,MHpE as well as DMH produced minimal to mild liverchanges such as vesiculation of nuclei and increase incytoplasmic homogeneity. However, in the absence ofsigns of necrosis or elevated level of aspartate amino-transferase in the serum, the urinary ascorbic acid andhepatic enzyme changes and histopathological changescan be considered as mild metabolic responses.
Another biochemical change shared by the three testcompounds and DMH was a dose-related decrease inliver TBARS levels which measured malondialdehydeproduced from lipid peroxidation. While an elevatedTBARS level indicates increased oxidative stress, thesignificance of a decreased level was unclear. TBARSmeasurements are subject to interferences by nonlipid-related materials and fatty peroxide-derived decompo-sition products other than malondialdehyde (Janero,1990; Moore and Roberts, 1998) and the results shouldbe interpreted with care. Other biomarkers of oxidationsuch as liver and serum carbonyls, a biomarker of pro-tein oxidation by reactive oxygen species (Stadtmanand Bertlett, 1998), and liver GSH levels were not sig-nificantly altered by treatment. Taken together, thesedata suggested that none of the four ethers is likely tob
edi MHb E.I of0 int MEf l tox-i al thes mice lare uri-n sl alt Ai nis-t a
ith the observed dysthrombopoiesis. In the pretudy, the DMH group also had the lowest platelet colthough the difference did not reach a statisticallyificant level.
Although all treated animals exhibited minimegree of changes in the thyroid, DMH appeareroduce more prominent changes. For example,
he DMH-treated animals had reduced colloid dennd the presence of papillary proliferation of the follar epithelium, which was indicative of a mild degf hyperplasia. However, in the absence of signifiodulation in serum thyroxin (T4) and triiodothyrine (T3), the thyroid effects may be adaptiveeversible.
Urinary ascorbic acid, a biomarker of hepaesponse to xenobiotics (Burns et al., 1960; Poon et a994) was elevated in all four ethers at 200 mg/kg, sesting that the hepatic glucuronic acid pathway, w
e a strong inducer of oxidative stress.A significant finding was that MAA was elevat
n the urine and plasma of animals treated with Dut not in animals treated with BE, EHxE and MHpt is interesting to note that the mean plasma MAA.34 mM is within the range of 0.1–1 mM reported
he blood of pregnant rats exposed to 50 ppm EGMor 5 days, a regimen that produced developmentacity (Gargas et al., 2000). Furthermore, the plasmevel of MAA appeared to be positively related toeverity of two separate toxic expressions, i.e. thyffect in terms of relative thymic weight, and testicuffects in terms of relative testicular weight andary creatine/creatinine ratio (Fig. 4). Such correlation
end support to the thesis that MAA is the proximoxic metabolite of DMH and that a high level of MAs associated with multiple adverse effects. Admiration of MAA is, of course, known to produced
110 R. Poon et al. / Toxicology 214 (2005) 99–112
multitude of adverse effects in rodents that target thetestes (Foster et al., 1984, 1987), the immune system(Miller et al., 1982; Smialowicz et al., 1992a,b), bonemarrow and haematopoietic system (Miller et al., 1982;Valentine et al., 1999), and fetal development (Brownet al., 1984; Sleet et al., 1996).
DMH is structurally similar to a class of glycolethers that include EGMME, EGDME, and DGDME(Fig. 1) which also elicit toxic expressions similar tothose produced by MAA. So far, MAA has been shownto be present in animals treated with ethylene glycolmonomethyl ether (Miller et al., 1983; Gargas et al.,2000; Hays et al., 2000), diethyleneglycol dimethylether (Cheever et al., 1988; Daniel et al., 1991; Richardset al., 1993), and now in DMH. However, the detectionof MAA raised the question of the nature of metabolicreactions that ultimately convert DMH to MAA. For acompound such as EGMME, the metabolite conversionto MAA is simply through oxidation reactions medi-ated by dehydrogenases (Miller et al., 1983). For morecomplex ether such as DGDME, cleavage of the centralether bond to EGMME followed by enzyme catalyzedoxidation to MAA was postulated to be the mechanism(Richards et al., 1993; Cheever et al., 1988). For DMH,an oxidative cleavage of the appropriate carbon–carbonbond in the hexane skeleton has to take place prior to theproduction of MAA via dehydrogenases. Further workis needed to investigate the possible involvement ofcytochrome P-450 enzymes in such oxidative cleavage.
The structure of MHpE also raised anotherm p ato hoxyg thata uces ows Aw bio-c stic-u , thed oupm H.I bel xyc malli thed ur-t suchs
Finally, the four ethers shared a common charac-teristic in that they did not appear to affect the kid-neys or the lungs as judged by the absence of notablehistopathological changes. The lack of a kidney effectwas supported by biochemical findings such as normalserum creatinine and urinary NAGA and protein levels.Unaltered bronchoalveolar levels of protein and NAGAalso pointed to a lack of pulmonary effects.
In summary, the toxicity data indicated that, at thedose levels tested, BE, EHxE and MHpE were not tes-ticular or thymic toxins. They shared some treatment-related effects with DMH in that all produced hep-atic, thyroid and bone marrow changes that were mildand likely adaptive in nature. The lack of testicularand thymic toxicity is likely related to an absence ofincrease in the level of the toxic metabolite MAA. Incontrast, the MAA level was significantly elevated inthe DMH group and its concentration appeared to becorrelated with the testicular and thymic effects. Theroute by which DMH is metabolized to MAA remainsto be elucidated.
Acknowledgements
The authors wish to thank A. Yagminas for profes-sional advice, B. Nadeau, A. McMahon, A. Lee, T.Hoeksma, S. Masson, Y. Dirieh, L. Overduin and RonStrathern for technical assistance. This work was sup-p ergyR
R
B of
B anta,he-guishhar-
B yl-in
69–
B .R.,of
col.
etabolite-related question. With a methoxy groune end of the carbon chain as compared to metroups at both ends of DMH, it may be postulatednimals treated with high dose MHpE should prodome MAA, albeit at a lower level, and hence shome MAA-related toxic expressions. In fact, MAas not significantly elevated and histological andhemical (creatine/creatinine ratio) evidence of telar damages was not present. On the other handecreased thymic weight in the high dose MHpE gray point to some subtle effects similar to that of DM
t is possible that monomethoxy compounds mayess effectively metabolized to MAA than dimethoompounds. It is also possible that there was a sncrease in MAA but at a level that was belowetection limit of the present HPLC procedure. F
her studies of similar ethers are needed to exploretructure activity relationships.
orted in part by the Federal Programme on Enesearch and Development.
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