Interactive effect of combined exposure to ethylene glycol ethers and ethanol on hematological parameters in rats

doi:10.4236/health.2010.29155

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Vol.2, No.9, 1054-1064 (2010) Health

doi:10.4236/health.2010.29155

Interactive effect of combined exposure to ethylene

glycol ethers and ethanol on hematological

parameters in rats

Andrzej Starek1, Katarzyna Miranowicz-Dzierżawska2*, Beata Starek-Świechowicz1

1Department of Biochemical Toxicology, Medical College, Jagiellonian University, Krakow, Poland

2Department of Chemical and Aerosol Hazards, Laboratory of Toxicology, Central Institute for Labour Protection—National Research

Institute, Warsaw, Poland; *Corresponding Author: kamir@ciop.pl

Received 23 April 2010; revised 14 May 2010; accepted 16 May 2010.

ABSTRACT

The study of the interaction of three glycol ethers,

i.e. 2-methoxyethanol (ME), 2-ethoxyethanol (EE)

and 2-butoxyethanol (BE) administered subcu-

taneously for 4 weeks and ethanol simultane-

ously given as 10% w/v solution for drinking in

male rats, was carried out from a toxicodynamic

point of view. Administered alone, ME (2.5 and

5.0 mM/kg), EE (2.5 and 5.0 mM/kg) or BE (0.75

and 1.25 mM/kg) resulted in a decrease of red

blood cells (RBC), packed cell volumes (PCV),

and hemoglobin concentration (HGB), as well as

an increase in mean corpuscular volume (MCV)

and reticulocyte count (Ret). In the rats co-ex-

posed to ethanol and EGAEs, a significantly less

pronounced hematological changes in compa-

rison with animal exposed to these ethers alone

were seen. The rats simultaneously exposed to

ethanol and both ME and EE at the lower dose

demonstrated mainly protection from the al-

terations in leukocyte system. In contrast, in the

rats which consumed ethanol and were simul-

taneously treated with the higher dose of ME or

EE (5.0 mM/kg) the amelioration of same hema-

tological parameters were displayed. The intake

of ethanol along with BE treatment at both

doses resulted in markedly ameliorated hema-

tological parameters, compared to those which

were changed by BE alone. In conclusion, the

decrease of the hemolytic effects of EGAEs is

ethanol dependent. Ethanol is a substrate of

alcohol dehydrogenase (ADH), and affinity of

this enzyme to ethanol is greater than that to

glycol ethers. It is possible that ethanol results

in the change in EGAEs metabolism.

Keywords: Ethylene Glycol Alkyl Ethers; Ethanol;

Repeated Exposure; Toxicodynamic Interactions

1. INTRODUCTION

Ethylene glycol alkyl ethers (EGAEs), i.e., 2-methoxye-

thanol (ME), 2-ethoxyethanol (EE), and 2-butoxyethanol

(BE) are extensively used as water-miscible organic

solvents in industrial and household applications. Inges-

tion, inhalation, and/or dermal absorption of these com-

pounds may lead to adverse testicular, teratogenic and

hematological effects in animals and humans. These

ethers may cause local toxicity, i.e., skin irritation and

sensitization in susceptible humans [1] and also, sys-

temic toxicity. The primary systemic toxicity of these

chemicals in animals include reproductive, developmen-

tal and hematological effects [2-6]. Similar effects have

been observed in exposed workers. The hematological

toxicity of ME resulted in marrow depression, leuko-

penia, pancytopenia, and decreased red blood cells count

(RBC), hemoglobin concentration (HGB), and platelet

count [7,8]. Shih et al. [9] showed that the HGB, packed

cell volume (PCV), and RBC in male workers exposed

to ME for 2.6 years were significantly lower relative to

controls. Also, the frequency of anemia in the exposed

group (26.1%) was significantly higher, with respect to

their corresponding control group (3.2%). Moreover, RBC

was significantly negatively associated with air concen-

trations of ME, whereas HGB, PCV, and RBC were

negatively correlated with urinary concentrations of me-

thoxyacetic acid (MAA), a metabolite of ME. These

hematological effects were reversible, and they had re-

turned to normal after reduction in ME exposure [10].

Other survey of male shipyard painters exposed to

mixed solvents containing EE acetate suggests that this

compound might be toxic to bone marrow [11], whereas

in female workers chronically exposed to high concen-

trations of EE, average RBC and HGB levels were nor-

A. Starek et al. / HEALTH 2 (2010) 1054-1064

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mal [12]. In male workers exposed to low concentration

of BE, a statistically significant decrease in PCV value

and an increase in mean cell hemoglobin concentration

(MCHC) value were observed [13].

EGAEs can produce toxicity following oxidation to

the corresponding aldehyde and alkoxyacetic acid by

alcohol dehydrogenase (ADH; EC 1.1.1.1) and aldehyde

dehydrogenase (ALDH; EC 1.2.1.3), respectively. Both

the alkoxyacetaldehyde and alkoxyacetic acid metabolites

of the EGAEs are considered to be toxic agents [14,15].

The metabolic activation of EGAEs by ADH and

ALDH leads to the appearance of their toxic effects

[16,17] . Butoxyacetic acid (BAA), a metabolite of BE,

is thought to be a more efficacious hemolytic agent than

the parent compound in vitro, and causes similar hemo-

lytic changes in vivo [17,18].

The inhibition of ADH by pyrazole or 4-methylpyra-

zole is known to decrease the hemolytic effect of BE

[17,19] and the urinary excretion of BAA [16,17]. The

simultaneous administration of BE with ethanol,

n-propanol or n-butanol to rats almost totally inhibited

the hemolytic effect of this chemical, and decreased the

urinary excretion of BAA. In contrast, the co-adminis-

tration of ethanol with ME did not modify the urinary

excretion of MAA, but induced accumulation of this

metabolite in rats [20,21]. These data mainly arise from

acute experiments.

This study presents the results of an investigation into

hematological changes in peripheral blood of rats simul-

taneously exposed to EGAEs and ethanol for 28 days.

Ethanol and other primary alcohols are used in industrial

solvent compositions containing glycol ethers and are

metabolized by ADH and ALDH.

In addition, the excessive consumption of ethanol by a

large part of the workers may lead to interactions with

EGAEs.

2. MATERIALS AND METHODS

2.1. Chemicals

ME, EE, and BE were purchased from Sigma-Aldrich

Ltd, Poland. Other chemicals were obtained from POCh

(Poland). ME, EE, and BE solutions were prepared in

saline, immediately before dosing, and administered to

rats by subcutaneous injections in a fixed volume of 2.0

ml/kg body weight, regardless of a dose.

2.2. Experimental Animals

Experiments were performed on 12-week-old male Wis-

tar rats (Krf: (WI)WUBR), with an initial body weight of

319 ± 22.4 g, and obtained from Jagiellonian University

Faculty of Pharmacy Breeding Laboratory (Kraków, Po-

land). The animals were kept under standard laboratory

conditions (temperature 21 ± 2˚C; relative humidity 50 ±

10%) with a 12 h:12 h (light:dark) cycle and had free

access to drinking water free of ethanol or 10% w/v

ethanol solution, and standard pellet Murigran chow

(Agropol, Motycz, Poland) during the experimental pe-

riod.

2.3. Experimental Design

The rats were randomly divided into fourteen groups of

five animals each. The rats (six groups) had free access

to drinking water and were treated with ME, and EE at

doses of 2.5, and 5.0 mM/kg or BE at doses of 0.75, and

1.25 mM/kg, 5 days per week, for 4 weeks. Control rats

(one group) received drinking water at libitum.

An ethanol groups (six groups) had free access to an

aqueous solution of ethanol (10% w/v solution of recti-

fied spirit POLMOS, Poland) as the only drinking fluid

and were treated by subcutaneous injections with ME,

EE or BE at the doses mentioned above. Control rats

(one group) drank 10% w/v ethanol, but were not ex-

posed to ME, EE or BE.

The rats were observed daily and were weighed once

weekly, whereas the consumption of an aqueous solution

of ethanol and food was measured daily during the

whole experiment.

Before experiment, during exposure and after its ter-

mination, i.e., at 0, 4, 11, 18, and 29 day, blood samples

from the tail vein of rats were collected for hematologi-

cal analyses.

The study was accepted by the Local Ethical Com-

mittee for animal experiments in Kraków. Procedures

involving the animals and their care conformed to the

institutional guidelines, in compliance with national and

international laws and Guidelines for the Use of Animals

in Biomedical Research.

2.4. Hematological Analyses

Heparin-added whole blood samples, immediately after

collection, were used for hematological analyses. RBC,

PCV, mean corpuscular volume (MCV), HGB, MCHC,

and mean cell hemoglobin (MCH) were analyzed by

means of a COBAS MICROS (Roche, Palo Alto, CA,

USA) analyzer. Reticulocyte count (Ret) was evaluated

after staining blood samples (without anticoagulant) with

brilliant-cresol blue. White blood cells (WBC) were

counted by a hemacytometer after diluting the fresh

blood samples (without anticoagulant) by Türk’s reagent

solution. The differential white cell count was evaluated

after Pappenheim-stained blood films.

Hematological analyses were systematically checked

by means of standard human blood CBC-3D Hematol-

ogy Control (R&D System Inc., Minneapolis. MN, USA).

A. Starek et al. / HEALTH 2 (2010) 1054-1064

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A day-to-day precision of RBC, PCV, and MCV meas-

urement (n = 30) in blood was 4.2, 4.5, and 4,4%, re-

spectively.

2.5. Statistical Analyses

Results are expressed as the mean ± SD. Data were ana-

lyzed by two-way analysis of variance with repeated

measurements on one factor and evaluation of simple

effects (MANOVA). The analysis was performed with

the SPSS 12.0 statistical packet (SPSS Inc., Chicago, IL,

USA). For comparison of exposed groups with control

group and exposed groups to ME, EE or BE alone with

groups simultaneously treated with these compounds and

ethanol at each point of time, one-way analysis of vari-

ance (ANOVA) followed by Dunnett test was used.

Probabilities lower than 0.05 were considered signifi-

cant.

3. RESULTS

Ethanol intakes in the ethanol alone and co-exposed to

ethanol and EGAEs groups were similar, when ex-

pressed in mM/kg b.w./24 h (Table 1).

Control rats consumed in average 59.6 ± 9.3 g/kg

b.w./24 h of Murigran food during the 4 week experi-

ment. The food consumption in the ethanol group and in

the co-exposed to ethanol and ME at a dose of 2.5

mM/kg group was similar to that of the control rats. In

other co-exposed groups, simultaneously treated with

ethanol and EGAEs, the food intake was significantly

diminished by mean 31% in comparison to the control

group (Table 1).

Body weight gain in both control and ethanol-drinking

groups was similar. An increase in the body weight, at

the end of exposure, in ethanol-drinking rats and simul-

taneously treated with ME, and EE at a dose of 2.5

mM/kg or exposed to BE at a dose of 0.75 mM/kg was

significantly lower in comparison to both control and

ethanol groups. The rats co-exposed to ethanol and ME

or EE at a dose of 5.0 mM/kg and BE at a dose of 1.25

mM/kg lost weight by 42.7 ± 5.1, 28.0 ± 6.2, and 11.0 ±

0.6 g, respectively (Table 1).

3.1. Hematological Changes

The consumption of ethanol alone for 4 weeks had no

effect on hematological parameters. ME administration

at doses of 2.5 and 5.0 mM/kg resulted in a decrease of

RBC, PCV, and HGB that occurred from 11th day of

exposure to its termination and in an increase in the Ret

exclusively at the end of exposure. At the end of ex-

periment, i.e., on day 29, the greatest changes of RBC,

PCV, HGB, and Ret were observed (Figure 1). These

hematological parameters demonstrated dose, both dose

and time, and time dependence.

In the rats co-exposed to ethanol and ME, signifi-

cantly less pronounced hematological changes were seen,

mainly in HGB, PCV and MCH (Figure 1).

Hematological changes in the peripheral blood of rats

treated with EE alone were less pronounced than those

after ME administration. The lower dose of this com-

pound (2.5 mM/kg) resulted only in a decrease of HGB

and an increase of MCV on days 11 and 18 of experi-

ment. The higher dose of EE (5.0 mM/kg) led to a sig-

nificant decrease in RBC, PCV, HGB, as well as to an

increase in MCV and Ret for most of the exposure time.

These hematological changes demonstrated dose de-

pendence and both dose and time, and time independ-

ence (Figure 2).

In the rats simultaneously treated with EE at the

higher dose (5.0 mM/kg) and ethanol, RBC, PCV, and

Table 1. Effects of ethylene glycol alkyl ethers on an average ethanol and food consumption and body weight gain in rats.

Group Ethanol intake

(mM/kg b.w./24 h)

Food intake

(g/kg b.w./24 h)

Body weight gain

(g/4 weeks)

Control1 59.6 ± 9.3 +24.3 ± 0.9

10% Ethanol 123.1 ± 18,9 52.3 ± 6.2 +23.0 ± 0.8

ME (2.5 mM/kg) + 10% ethanol 90.3 ± 20,0 55.5 ± 8.7 +2.4 ± 0,2*†

ME (5.0 mM/kg) + 10% ethanol 115.0 ± 23.9 36.6 ± 3.9*† –42.7 ± 5.1*†

EE (2.5 mM/kg) + 10% ethanol 136.7 ± 1.9 42.7 ± 3.4*† +4.0 ± 0.4*†

EE (5.0 mM/kg) + 10% ethanol 145.3 ± 16.0 39.2 ± 5.8*† –28.0 ± 6.2*†

BE (0,75 mM/kg) + 10% ethanol 130.0 ± 3.1 42.3 ± 3.7*† +4.0 ± 0.1*†

BE (1,25 mM/kg) + 10% ethanol 124.0 ± 6.2 43.2 ± 4.1*† –11.0 ± 0.6*†

1The rats had free access to drinking water. Values are the means ± SD of 5 animals/group. p < 0.05 compared to control (*) and ethanol (†) group.

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Figure 1. Effects of ethanol (EtOH) consumption on RBC (a), PCV (b), HGB (c), MCH (d) and Ret (e) values of the

peripheral blood at the designated time points of male rats treated with methoxyethanol (ME) at doses 2.5 mM/kg b.w.

or 5.0 mM/kg b.w. The values are means  SD of five rats, *—P  0,05 significantly different from control rats; †—

P  0,05 significantly different from rats treated with ME alone.

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Figure 2. Effects of ethanol (EtOH) consumption on RBC (a), PCV (b), HGB (c), MCV (d) and Ret (e)

values of the peripheral blood at the designated time points of male rats treated with ethoxyethanol (EE) at

doses 2.5 mM/kg b.w. or 5.0 mM/kg b.w. The values are means  SD of five rats, *—P  0.05 significantly

different from control rats; †—P  0.05 significantly different from rats treated with EE alone.

A. Starek et al. / HEALTH 2 (2010) 1054-1064

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HGB at day 4, and HGB at day 11, were significantly

higher than in the rats exposed to EE alone. These pa-

rameters did not differ in comparison to the control

group (Figure 2).

BE administration at doses of 0.75 and 1.25 mM/kg

resulted in a decrease in RBC, PCV, and HGB and an

increase in MCV, and Ret. The greatest changes of RBC,

PCV, and HGB were observed at day 4 of experiment,

whereas other hematological alterations (MCV, and Ret)

showed a maximum on day 11 of the exposure. The

changes in RBC, MCV, and Ret during exposure period

were persistent, whereas alterations in PCV, and HGB

independently on duration of exposure, were reversible

(Figure 3).

In the rats co-exposed to BE and ethanol, the values of

RBC, PCV, and HGB were significantly higher, whereas

MCV, and Ret were markedly lower in comparison to

the rats treated with BE alone (Figure 3). Some of he-

matological parameters (HGB, MCV, and MCHC) in

rats exposed to BE at a lower dose (0.75 mM/kg) and

ethanol were similar to those in the control group.

3.2. Leukocyte Alterations

The leukocyte counts were markedly reduced at each

dose of ME from the 4th day of exposure to its termina-

tion. These alterations were caused by a decrease mainly

in the number of lymphocytes. Also, reductions in num-

ber of neutrophils in rats treated with ME at a dose of

5.0 mM/kg at days 11 and 18 of the experiment were

observed. Both leukocyte and lymphocyte alterations at

a dose of 5.0 mM/kg demonstrated dose, dose and time,

and time dependence (Figure 4).

The rats simultaneously exposed to ethanol and the

lower dose of ME (2.5 mM/kg) showed a lack of changes

in both leukocyte and lymphocyte counts in comparison

to control group at days 11, and 18 of the experiment.

The number of lymphocytes in those animals at days 18

and 29 of the experiment was significantly lower than in

the control group, but statistically higher in comparison

to the rats treated with ME alone. In the rats co-exposed

to ethanol and the higher dose of ME (5.0 mM/kg), the

leukocyte and lymphocyte counts were significantly di-

minished in comparison to the control group, but very

similar as in the group exposed to ME alone. Also, the

reduction of the number of neutrophils at days 11, 18,

and 29 was observed (Figure 4).

No significant leukocyte and lymphocyte alterations

in rats exposed to EE at a dose of 2.5 mM/kg were ob-

served. In the rats treated with EE alone at the higher

dose (5.0 mM/kg) the leukocyte counts at day 4 and 18

of the experiment were statistically lower than in the

control group. The number of lymphocytes in these rats

was reduced in comparison to the control group during

the whole of exposure period (Figure 5).

The number of both leukocytes and lymphocytes in

the rats co-exposed to EE at a dose of 2.5 mM/kg and

ethanol only at day 4 of the experiment was significantly

higher in comparison to group treated with EE alone. In

the rats simultaneously exposed to ethanol and EE at the

higher dose (5.0 mM/kg) the leukocyte counts on day 4,

and the number of lymphocytes on days 4, and 29 were

significantly lower than in the control group (Figure 5).

BE alone had no effect on leukocyte and lymphocyte

counts in peripheral blood in rats treated at the dose of

0.75 mM/kg and 1.25 mM/kg for 28 days.

In the rats simultaneously exposed to ethanol and BE

at the dose of 0.75 mM/kg and 1.25 mM/kg the number

of both leukocytes and lymphocytes in peripheral blood

were similar as in the control group.

4. DISCUSSION

The aim of the present work was to assess the effect of

ethanol drinking on hemolytic action of EGAEs in rats.

The experimental protocol applied in this experiment

aimed at the constitution of a model of conditions that

may take place in human life.

The treatment with ethanol may be tantamount to its

misuse in man. The daily consumption of ethanol in the

rats drinking 10% (w/v) water solution of ethanol was

equivalent to about 0.7 l/day of 40% vodka in men [22].

Since the rate of ethanol oxidation in rats (0.3 g/kg/h) is

three times faster than in humans, these animals needed

a higher dose of ethanol to produce comparable toxic

effects.

The rats simultaneously treated with EGAEs and

ethanol consumed similar quantity of ethanol as in the

control group. Food intake in these animals was dimin-

ished in each experimental group in relation to both con-

trol and ethanol groups. The reduction in food intake

was most pronounced at the highest dose of ME, EE, and

BE, i.e., 5.0, 5.0, and 1.25 mM/kg, respectively. The

growth retardation in rats simultaneously exposed to

EGAEs and ethanol was observed. It was most likely to

be caused by the reduced food consumption observed in

these animals. Although ethanol has been reported to

cause anorexia and weight loss [23,24], it seems that

EGAEs alone may be one of the main reasons for retar-

dation of body weight gain. The body weight alterations

observed previously in rats exposed to EGAEs alone [6]

seem to confirm of this suggestion.

In the present study, it was found that subcutaneous

repeated administration of each of three EGAEs led to

distinct hematological alterations. These alterations were

evidenced by reduction in RBC, PCV, and HGB, and

also by an increase in MCV value and Ret in peripheral

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Figure 3. Effects of ethanol (EtOH) consumption on RBC (a), PCV (b), HGB (c), MCV (d) and Ret (e) values of the

peripheral blood at the designated time points of male rats treated with buthoxyethanol (BE) at doses 0.75 mM/kg b.w.

or 1.25 mM/kg b.w. The values are means  SD of five rats, *—P  0,05 significantly different from control rats; †—

P  0.05 significantly different from rats treated with BE alone.

A. Starek et al. / HEALTH 2 (2010) 1054-1064

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Figure 4. Effects of ethanol (EtOH) consumption on WBC (a), NEU (b) and LYM (c) values of the peripheral

blood at the designated time points of male rats treated with methoxyethanol (ME) at doses 2.5 mM/kg b.w. or

5.0 mM/kg b.w. The values are means  SD of five rats, *—P  0,05 significantly different from control rats;

†—P  0,05 significantly different from rats treated with ME alone.

blood. While in rats treated with ME these changes were

strongly pronounced and progressively increased with

exposure time beginning from the day 11, those in ani-

mals treated with EE were less pronounced and rather

persisted at low constant level for the whole exposure

period. On the contrary, the rats treated with BE demon-

strated the distinct intravascular hemolysis resulted in

hemolytic anemia at the beginning of exposure (on day

4). Independently of exposure duration, these alterations

were regressed, although the decrease in RBC and the

increase in MCV were more persistent, probably due to

the selective hemolysis of the aged erythrocytes [25],

leaving a population of young red cells. Hemoglobinuria

observed only in the first day of exposure to BE at dose

of 1.25 mM/kg seems to confirm of this suggestion. The

various hematological changes observed during expo-

sure to EGAEs are typical of hemolytic anemia with an

associated reticulocytosis and hyperplasia of both bone

marrow (erythroid elements) and spleen (extramedullary

hemopoiesis) [26].

The results presented in this paper confirm previous

observations that continued exposure to BE, contrary to

ME and EE, resulted in significantly less pronounced

hematological changes [6]. While the majority of hema-

tological effects were dramatic at the beginning of the

exposure, later these alterations clearly regressed despite

continued weekly exposure to these compounds. It was

suggested that the gradual recovery from the haemolytic

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Figure 5. Effects of ethanol (EtOH) consumption on WBC (a) and LYM (b) values of the peripheral blood at the

designated time points of male rats treated with ethoxyethanol (EE) at doses 2.5 mM/kg b.w. or 5.0 mM/kg b.w.

The values are means  SD of five rats, *—P  0.05 significantly different from control rats; †—P  0.05 sig-

nificantly different from rats treated with EE alone.

anemia may be associated with tolerance development to

the hemolytic effect of BE. This tolerance was character-

rized by a progressive removal of hematological changes,

manifested in an increase of RBC, PCV, HGB, and a

decrease in Ret value in peripheral blood. The tolerance

to BE-induced hemolytic anemia was also observed in

other studies [27,28]. Moreover, our studies demonstra-

ted that the effects of repeated exposure for 28 days to

BE were less pronounced than the effects of single doses

of this ether at the same or comparable doses [29,30].

The changes in the leukocyte system of rats were

produced by two of three EGAEs, but there were marked

quantitative differences in the responses. While ME

strongly suppressed this system causing leukopenia,

lymphocytopenia, and neutrocytopenia, EE at the higher

dose (5.0 mmol/kg) only, exerted the middle inhibitory

effect resulted in the occasional reduction of leukocytes

with a simultaneous decrease in the number of lympho-

cytes at each time point of the exposure. In contrast, BE

caused no changes in the leukocyte system. These results

are inconsistent with the data of Grant et al. [26] which

observed inhibitory effects of ME and BE on leukocyte

system in rats. This inconsistency concerning BE only,

may be due to considerably higher doses of this com-

pound administered to rats, reported in the paper cited.

The literature data [26,31] indicate that ME is immu-

notoxic. Dermal exposure of rats to this compound at

dose levels in a range of 2-16 mM/kg/day for four con-

secutive days produce droplets in thymus and spleen

weight, enhanced the lymphoproliferative responses to

mitogens, and a reduction in the antibody plaque-forming

cell response to either trinitrophenyl-lipopolysaccharide

or sheep RBC. Miller et al. [32] observed lymphoid tis-

sue atrophy after inhalation of ME in male New Zealand

white rabbits. Also Smialowicz et al. [33,34] reported a

marked immunosuppressive effects of ME and both

MAA and methoxyacetaldehyde, the metabolites of ME,

on thymus weight and lymphoproliferative functions in

rats.

There is still too little knowledge on the effects of

ethanol on EGAEs-toxicity, especially on hematological

changes. Morel et al. [21] did not observed the effects of

three aliphatic alcohols, i.e., ethanol, n-propanol, and

n-butanol, at dose of 10 or 30 mmol/kg, on the urinary

creatine/creatinine ratio, the testicular toxicity or the 24

h urinary excretion of MAA in rats after simultaneous

treatment with a single dose of ME (10 mM/kg) by ga-

vage. On the other hand, the simultaneous administration

of 30 mM/kg of above mentioned alcohols almost totally

inhibited the hemolytic effect of BE (5 or 1 mM/kg), and

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reduced the urinary excretion of BAA, metabolite of this

compound, by 31-43%. It was suggested that competi-

tive inhibition of ADH by alcohols results in the change

in BE metabolism.

The results of the present study indicate that ethanol

alone consumption did not have any effect on examined

hematological parameters. However, ethanol intake along

with EGAEs, only partially protected the rats against

hemolytic effects and the alterations in leukocyte system

induced by these ethers. The preventive effect was seen

at both lower and higher doses of ME (2.5 and 5.0

mM/kg) and BE (0.75 and 1.25 mM/kg), as well as at the

higher dose of EE (5.0 mM/kg), while in rats simultane-

ously exposed to ethanol and both ME and EE at the

lower dose (2.5 mM/kg), mainly protection from the

alteration in leukocyte system was observed. In contrast,

the rats which consumed ethanol and simultaneously

were treated with the higher dose of ME or EE (5.0

mM/kg), demonstrated the amelioration of these hema-

tological parameters (RBC, PCV, and HGB) in relation

to the animals exposed to these compounds alone. On

the other hand, ethanol intake along with BE treatment

(both 0.75 and 1.25 mM/kg) markedly ameliorated he-

matological parameters, especially RBC, PCV, HGB,

MCV, and Ret in comparison with the group exposed to

this ether alone.

The results presented in this paper clearly demonstrate

that ethanol modifies the hematological effects of con-

tinued exposure to EGAEs. The explanation the biologi-

cal basis of the interactions between ethanol and EGAEs

may be related to the metabolism of these compounds.

The glycol ethers, especially EE, BE, and 2-phenoxye-

thanol, are metabolized in vitro by rat hepatic and cuta-

neous cytosolic preparations in the reaction involving

ADH and ALDH in both tissues [35]. Hepatic cytosol

metabolizes ethanol in preference to intermediate chain-

length EGAEs, whereas the skin cytosol preferentially

metabolizes the glycol ethers. It was found that EGAEs

oxidation is performed predominantly by ADH3, and

ALDH1 isoenzymes. Repeated exposure to EGAEs re-

sults in induction of these enzymes. The rates of ADH

oxidation by rat liver cytosol were the greatest for etha-

nol followed by EE and BE. In contrast, the order of

metabolism by rat skin cytosolic fraction was changed to

BE > EE > ethanol [35]. Although skin contains en-

zymes that have the capacity to biotransformation EGAEs

localized in the basal layer of the epidermis [36], the

physicochemical properties of these compounds result in

rapid penetration and distinct reduction in dermal me-

tabolism during their percutaneous absorption [37]. Thus,

EGAEs are mainly metabolized in the liver similarly as

ethanol.

In conclusion, the decrease of the hemolytic effects due

to EGAEs is ethanol dependent. Ethanol is a substrate of

ADH, and the affinity of this enzyme is higher than

those of glycol ethers. It is possible that ethanol results

in the change in EGAEs metabolism. The difference

between EGAEs metabolism may be one of the reasons

for the differences observed between ME, EE, and BE in

the interaction of ethanol.

5. ACKNOWLEDGEMENTS

This paper has been prepared on the basis of the results of a research

task 2.R.05 carried out within the scope of the first stage of the Na-

tional Programme “Improvement of safety and working conditions”

supported in 2008-2010—within the scope of research and develop-

ment – by the Ministry of Science and Higher Education.

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