Assessment of the impact of methyl tertiary butyl ether (MTBE) contaminated drinking water on blood hematology.

INTRODUCTION

MTBE has been used in the United States (U.S.) gasoline since 1979, in low levels (between 0.5 to 3.5% w/v) to replace lead. Since 1992, MTBE has been used at high concentrations in gasoline (15% w/v) to fulfill the oxygenate requirements set in some U.S. States by Congress in the 1990 Clean Air Act Amendments. In 1994, MTBE was the 18th most important chemical produced in the U.S.A. In 1999, over 200,000 barrels per day was produced in the U.S., which is almost exclusively used as a fuel additive in motor gasoline (Gillner, 1998).

In January 2001, leaded car fuel in Saudi Arabia was replaced by unleaded fuel and was consequently distributed by all gas stations across the Kingdom. This move came as a result of the mounting evidence that lead causes deleterious effects on health and environment. A synthetic organic substance, called methyl tertiary-butyl ether (MTBE), was produced in the Kingdom by SABIC and delivered to Saudi Aramco for distribution. MTBE is also produced in other Arabian Gulf States in very large quantities (EIA, 2002).

MTBE is one of a group of chemicals commonly known as "oxygenates" because they raise the oxygen content of gasoline, and therefore increase its octane number and reduce automotive emissions, such as carbon monoxide and hydrocarbons. The use of oxygenated fuels such as MTBE is anticipated to increase over the next decades (Costantini, 1993).

Yet, the issue of MTBE has been controversial, in part because of concerns about potential inhalation health effects and more recently because of an added concern about MTBE-contaminated drinking water (EPA, 2004).

The U.S. Environmental Protection Agency (EPA) now requires monitoring of MTBE and other oxygenate compounds in ground water at leaking underground storage tank sites nationwide since environmental officials classify this additive as a hazardous substance (EPA, 2004). To further complicate the problem, the major metabolites of MTBE exposure in humans are methanol, formaldehyde and tertiary butyl alcohol (TBA) produced as a result of microsomal oxidation by cytochrome P-450 enzymes (CYP's) (Hutcheon et al., 1996). These active metabolites are known to be toxic to humans (Casarett & Doull, 2001).

Despite the growing concerns over the use of MTBE, few biochemical data on this chemical have been published. Most studies on health issues concentrated on the neurotoxicological aspects (Daughtrey et al., 1997), genotoxicity (Kado et al., 1998), mutagenecity, and carcinogenecity (Zhou et al., 2000), the induction of programmed cell death (apoptosis) and inhibition of cell cycle progression (Vojdani et al., 1997).

Methyl tertiary butyl ether (MTBE) has a chemical formula [C.sub.5][H.sub.12]O, and a molecular weight of 88.15. The structural formula of MTBE is shown in (Fig.1). It is a clear, colorless, flammable liquid with a distinctive characteristic ethereal odor. Its density is 0.741, boiling point 55.2, melting point -109, flash point -28, vapor pressure 245, specific gravity 0.74, it is soluble in alcohol and ether, solubility in water of 4.8 g/100g. No information is availble about its pH. Other names of MTBE are 2-Methoxy-2-methylpropane; tertiary butyl methyl ether; Methyl, 1-dimethyl ethyl ether (Galvis, 2000).

[FIGURE 1 OMITTED]

MTBE is prepared by the acid-catalyzed addition of methanol to 2-methylpropene (Galvis, 2000), as follows:

[FORMULA NOT REPRODUCIBLE IN ASCII]

Humans are exposed to MTBE on a daily basis. For example, through touching the skin or breathing contaminated air while pumping gasoline, or breathing exhaust fumes while driving their cars, and breathing MTBE-contaminated air near highways or in cities. They are also exposed to it, while drinking, swimming, or showering in water that has been contaminated with MTBE. MTBE is used also as a therapeutic drug to dissolve gallstones (ATSDR, 2001). Therefore, this study focused on the possible health hazards of MTBE in drinking water as manifested by changes on blood hematology. The study tries also to draw a quantitative relationship between MTBE concentration and changes of blood hematology.

MATERIALS AND METHODS

Animals

The source organs in this study was as follows: 120 male Wistar rats that had a mean initial body weight of 139.14 [+ or -] 1.76g with 6 weeks of age. They were randomly selected from a rat colony bred from animals obtained in 1976 from Olac Ltd, U.K. in the Experimental Animal Unit of King Fahad Medical Research Center. They were housed in an air-conditioned room with indoor temperature maintained at 24[degrees]C and exposed to a 12hr dark/light cycle. Animals were kept in plastic cages, on wood shaving bedding. They were fed on standard animal feed produced by Grain, Silos and Flour Mills Organization, Western Province, Saudi Arabia. The animal house was approved and licensed. Rats were then divided into five groups, forty rats for control group, and each of the other four groups had twenty rats, and every five rats were kept in one cage. Rats were individually weighed at the beginning of the study and at the end of the experiment period (60-days).

MTBE Sample

MTBE sample was provided by Saudi-Aramco, Jeddah, Saudi-Arabia and was used as it is, without further purification.

Drinking Water

Drinking water containing different concentrations of MTBE was supplied daily (0.0, 1,000, 1,500, 2,000, and 2,500 ppm) for 60-days. Tap water mixed with MTBE was available for the rats 24hr a day. Water was replaced every 6 days. The concentration of MTBE in drinking water was calculated as shown in (Table 1).

The water under study was prepared by taking the proper amount of water in a beaker and placed on the balance, and adjusted to zero gram, then the required MTBE concentration was added. This procedure was used to prevent the evaporation of MTBE. The solution was then placed in a measured conical flask, that was filled up with water. Each cage was supplied with a certain concentration of MTBE ranging from 1,000 to 2,500 ppm.

Blood Chemistry

At the end of the experimental period (60 days) blood samples were collected from animals in EDTA tubes by cardiac puncture under diethyl ether anesthesia for complete blood count (CBC) analysis by COULTER GEN.STM System.

RESULTS

Blood of MTBE treated rats for 60 days, showed significant decrease in white blood cell (WBC) count ([10.sup.3]/[micro]l) at low MTBE concentrations (1,000, and 1,500 ppm) by 55.8% and 39.1% respectively. On the other hand, at higher MTBE concentrations (2,000, and 2,500 ppm), WBC counts increased (Fig. 2) by 23.3% and 18.4% respectively (Table 2).

[FIGURE 2 OMITTED]

There was no significant difference at all MTBE concentrations (1,000, 1,500, 2,000, and 2,500 ppm) in red blood cells (RBC) count (106/[micro]l), in platelets (Plt) count (106/[micro]l), and in hemoglobin (Hb) concentration (g/dl) in treated animals (Table 2).

The absolute numbers of neutrophils (NE) count ([10.sup.3]/[micro]l) in treated rats increased significantly at high MTBE concentrations (2,000, and 2,500 ppm) by 61.2, and 53.1% respectively, but at low MTBE concentrations (1,000, and 1,500 ppm) there was no significant effect on neutrophils (NE) count ([10.sup.3]/[micro]l) (Fig. 3) in treated animals (Table 3).

The absolute numbers of lymphocytes (LY) count ([10.sup.3]/[micro]l) in treated rats decreased significantly only at low MTBE concentration (1,000 ppm) by 36.2%, and increased significantly at high MTBE concentrations (2,000, and 2,500 ppm) by 25.0% and 24.4% respectively (Fig. 4). But at MTBE concentration 1,500 ppm, there was no significant effect on lymphocytes (LY) count ([10.sup.3]/[micro]l) in treated animals (Table 3).

On the other hand, no significant difference was noted in the absolute numbers of monocytes (MO), eosinophils (EO), and basophils (BA) counts ([10.sup.6]/[micro]l) at all MTBE concentrations (1,000, 1,500, 2,000, and 2,500 ppm) in treated animals (Table 3).

Mean corpuscular hemoglobin (MCH) (pg) in treated rats decreased significantly at low MTBE concentrations (1,000, and 1,500 ppm) by 8.6% and 6.2% respectively, and increased significantly at high MTBE concentrations (2,000, and 2,500 ppm) (Fig. 5) by 7.4% and 5.2% respectively (Table 4).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Mean corpuscular hemoglobin concentration (MCHC) (g/dl) in treated rats decreased significantly at low MTBE concentrations (1,000, and 1,500 ppm) by 9.2% and 7.4% respectively, and increased significantly at high MTBE concentrations (2,000, and 2,500 ppm) (Fig. 6) by 8.3% and 5.7% respectively (Table 4).

There was no significant difference in hematocrit (Hct) (%), red distribution width (RDW) (%), and in mean corpuscular volume (MCV) (fl) at all MTBE concentrations (1,000, 1,500, 2,000, and 2,500 ppm) in treated animals (Table 4).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

DISCUSSION

The data show that rats did not suffer any forms of anemia as a consequence of MTBE treatment. Thus, RBC (red blood cells) count was not changed at any concentration level of MTBE, nor hemoglobin (Hb) content. Moreover, other hemotologic parameters, related to anemic conditions, were not affected either, such as Hct (hematocrit), RDW (red distribution width), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration). These results are compatible with the current literature, which shows no evidence of MTBE induction of anemia in experimental animals (Feldman et al., 2000).

Other hematologic parameters show a biphasic pattern of increase and decrease of white blood cells (WBC) according to MTBE concentration. Thus, at lower MTBE concentrations (1,000, and 1,500 ppm) WBC count was reduced significantly while at higher concentrations (2,000, and 2,500 ppm) their counts were significantly elevated. This biphasic pattern of WBC count was positively correlated with a similar pattern of decrease and increase exhibited by neutrophils (NE) and lymphocytes (LY). Other types of white blood cells (eosinophils, basophils and monocytes) were not affected.

Therefore, we suggest that the biphasic effect of MTBE on white blood cells (WBC) is due mainly to changes in the numbers of neutrophils (NE) and lymphocytes (LY). Neutrophils (NE) constitute the major cellular component of the mammalian blood and play a key role in both innate and specific immunity. Lymphocytes (LY) on the other hand, are the chief cells of specific or acquired immunity (Fischbach & Dunning lll, 2003; Desal & Isa-Pratt, 2000; Besa et al., 1993). These results are compatible with the case of hepatic injury flowed by inflammation lead to both neutrophilia and lymphocytosis which is called leukocytosis. Leukocytosis is a common feature of inflammatory reaction (Robbins et al., 1994).

REFERENCES

(1.) ATSDR (Agency for Toxic Substances and Disease Registry) 2001.. Methyl tertiary butyl ether (MTBE).

(2.) Besa, E., Catalono, P., Kanta, J., and Jefferies, L. 1993. Hematology.

(3.) Casarett, L., and Doull, J. 2001. Toxicology: The Basic Science of Poisons Collier Macmillan Canada, Ltd, Toronto, London.

(4.) Costantini, M. 1993. Health effects of oxygenated fuels. Environ Health perspect., Suppl. 6:151-60.

(5.) Daughtrey, W., Gill, M., Pritts, I., Douglas, J., Kneiss, J., and Andrews, L. 1997. Neurotoxicological evaluation of methyl tertiary butyl ether in rats. J Appl Toxicol, Suppl. 1:S57-64.

(6.) Desal, S., and Isa-Pratt, S. 2000. Clinical's Guide to Laboratory Medicine.

(7.) EIA (Energy Information Administration) 2002. Saudi Arabia: environmental issues.

(8.) EPA (Environmental Protection Agency) 2004. MTBE (Methyl tertiary butyl ether) and under ground storage tanks.

(9.) Feldman, Zinkl, and Jain 2000. Veterinary Hematology. 5th edition.

(10.) Fischbach, F., and Dunninglll, M. 2003. A Manual of Laboratory and Diagnostic Tests. 7th edition.

(11.) Galvis, O. 2000. Methyl tert-butyl ether (MTBE), Environmental Health Criteria.

(12.) Gillner, M. 1998. Methyl tertiary butyl ether. Environmental Health Criteria, 206.

(13.) Hutcheon, D., Arnold, J., Ten Hove, W., and Boyle, J. 1996. Disposition, metabolism, and toxicity of methyl tertiary butyl ether, an oxygenate for reformulated gazoline. Toxicol Environ Health, 47(5):453-64.

(14.) Kado, N., Kuzmicky, P., Loarca-Pina, G., and Mumtaz, M. 1998. Genotoxicity testing of methyl tertiary butyl ether (MTBE) in the Salmonella microsuspension assay and mouse bone marrow micronucleus test. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 412:2:131-138.

(15.) Robbins, R., Cotran, R., and Kumar, V. 1994. Robbins Pathologic Basis of Disease. 5th edition.

(16.) Vojdani, A., Mordechai, E., and Brautbar, N. 1997. Abnormal apoptosis and cell cycle progression in humans exposed to methyl tertiary butyl ether and benzene contaminating water. Hum Exp Toxicol, 16: 485-94.

(17.) Zhou, W., Yuan, D., Huang, G., Zhang, H., and Ye, S. 2000. Mutagenicity of methyl tertiary butyl ether. J Environ Pathol Toxicol Oncol, 19:35-9.

Wadiah Saleh Backer and Hanadi Ahmed Katouah

King Abdulaziz University, Biochemistry Department, Saudi Arabia. P.O. Box 42737, Jeddah 21551, Email:[e-mail]

Table 1: Concentration of MTBE in water.

                               Conc. of MTBE in water
Group no.      No. of rats          ppm         g/L

    1              40               0.0         0.0
    2              20             1,000         1.0
    3              20             1,500         1.5
    4              20             2,000         2.0
    5              20             2,500         2.5

Table 2: Effect of different MTBE concentrations on blood hematology of
rats (at day 60) *.

                            WBC                      RBC
                      (103/[micro]l)           (106/[micro]l)

       Control      6.86 [+ or -] 0.38       8.38 [+ or -] 0.19
                           n=23                     n=28

     1,000ppm       3.03 [+ or -] 0.55       8.22 [+ or -] 0.28
                            n=7                     n=10

Control Vs  P<               0                       NS
1,000ppm

      1,500ppm     4.18 [+ or -] 0.49       8.43 [+ or -] 0.22
                           n=10                     n=14

Control Vs  P<               0                       NS
1,500ppm

      2,000ppm    8.46 [+ or -] 0.34       8.08 [+ or -] 0.11
                           n=16                     n=19

Control Vs  P<             0.005                     NS
2,000ppm

     2,500ppm     8.12 [+ or -] 0.3        8.15 [+ or -] 0.1
                           n=16                     n=18

Control Vs  P<             0.019                     NS
2,500ppm

                            PLT                      Hb
                      (103/[micro]l)               (g/dl)

    Control         935.74 [+ or -] 34.36     13.56 [+ or -] 0.22
                           n=23                     n=28

   1,000ppm         899.33 [+ or -] 49.84     12.75 [+ or -] 0.2
                            n=6                      n=8

Control Vs  P<              NS                       NS
1,000ppm

   1,500ppm        938.75 [+ or -] 46.37     12.99 [+ or -] 0.33
                           n=12                     n=14

Control Vs  P<              NS                       NS
1,500ppm

    2,000ppm       945.71 [+ or -] 33.09     14.14 [+ or -] 0.16
                           n=18                     n=19


Control Vs  P<              NS                       NS
2,000ppm

     2,500ppm       947.61 [+ or -] 28.64     13.98 [+ or -] 0.17
                           n=18                     n=18

Control Vs  P<              NS                       NS
2,500ppm

Avearge [+ or -] SE; Significance (P) <0.05

* End of the experimental period

WBCs = white blood cells or leukocytes

RBCs = red blood cells or erythrocytes

Plt = platelets or thrombocytes

Hb = hemoglobin

Table 3: Effect of different MTBE concentrations on absolute number of
differential leukocytes ([10.sup.6]/[micro]l) of rats at day 60) *.

                            NE                       LY
                   ([10.sup.3]/[micro]l)    ([10.sup.3]/[micro]l)

Control             0.49 [+ or -] 0.07       5.69 [+ or -] 0.35
                           n=23                     n=26

1,000ppm             0.37 [+ or -] 0.1       3.63 [+ or -] 0.6
                            n=9                     n=10

Control    P<               NS                     0.005
  Vs
1,000ppm

1,500ppm            0.49 [+ or -] 0.08       5.59 [+ or -] 0.79
                           n=13                     n=13

Control    P<               NS                       NS
   Vs
1,500ppm

2,000ppm             0.79 [+ or -] 0.1       7.11 [+ or -] 0.42
                           n=19                     n=18

Control    P<              0.013                   0.013
   Vs
2,000ppm

2,500ppm            0.75 [+ or -] 0.09       7.08 [+ or -] 0.34
                           n=11                     n=18

Control    P<              0.04                     0.01
   Vs
2,500ppm

                            MO                       EO
                   ([10.sup.3]/[micro]l)    ([10.sup.3]/[micro]l)

Control             0.03 [+ or -] 0.01       0.03 [+ or -] 0.01
                           n=28                     n=28

1,000ppm            0.01 [+ or -] 0.01       0.03 [+ or -] 0.02
                           n=10                    n=10

Control    P<               NS                       NS
  Vs
1,000ppm

1,500ppm            0.01 [+ or -] 0.01       0.05 [+ or -] 0.02
                           n=14                     n=14

Control    P<               NS                       NS
   Vs
1,500ppm

2,000ppm            0.02 [+ or -] 0.01       0.02 [+ or -] 0.01
                           n=19                     n=19

Control    P<               NS                       NS
   Vs
2,000ppm

2,500ppm            0.03 [+ or -] 0.02       0.03 [+ or -] 0.01
                           n=18                     n=18

Control    P<               NS                       NS
   Vs
2,500ppm

                            BA
                   ([10.sup.3]/[micro]l)

Control             0.03 [+ or -] 0.01
                           n=28

1,000ppm            0.01 [+ or -] 0.01
                           n=10

Control    P<               NS
  Vs
1,000ppm

1,500ppm             0.0 [+ or -] 0.0
                           n=14

Control    P<               NS
   Vs
1,500ppm

2,000ppm            0.02 [+ or -] 0.01
                           n=19

Control    P<               NS
   Vs
2,000ppm

2,500ppm            0.02 [+ or -] 0.01
                           n=18

Control    P<               NS
   Vs
2,500ppm

Avearge [+ or -] SE; Significance (P) <0.05

* End of the experimental period

NE = neutrophils or granulocytes

LY = lymphocytes

MO = monocytes

EO = eosinophils

BA = basophils

Table 4: Effect of different MTBE concentrations on hematocrit (Hct)
(%), red distribution width (RDW) (%), mean corpuscular volume (MCV)
(fl), mean corpuscular hemoglobin (MCH) (pg), and mean corpuscular
hemoglobin concentration (MCHC) (g/dl) of blood hematology  of rats
(at day 60) *.

                           Hct                    RDW
                           (%)                    (%)

 Control           44.9 [+ or -] 0.94     13.53 [+ or -] 0.13
                          n=27                   n=28

1,000ppm           45.72 [+ or -] 0.94    13.32 [+ or -] 0.17
                           n=9                   n=10

Control     P<             NS                     NS
   Vs
1,000ppm

1,500ppm           44.51 [+ or -] 1.23    13.56 [+ or -] 0.18
                          n=14                   n=14

Control     P<             NS                     NS
   Vs
1,500ppm

2,000ppm           43.01 [+ or -] 0.49    14.06 [+ or -] 0.44
                          n=19                   n=19

Control     P<             NS                     NS
   Vs
2,000ppm

2,500ppm           43.62 [+ or -] 0.53    13.83 [+ or -] 0.14
                          n=17                   n=18

Control     P<             NS                     NS
   Vs
2,500ppm

                           MCV                    MCH
                          (fl)                   (pg)

 Control           53.58 [+ or -] 0.28    16.32 [+ or -] 0.29
                          n=28                   n=28

1,000ppm           54.03 [+ or -] 0.27    14.91 [+ or -] 0.12
                          n=10                   n=10

Control     P<             NS                     0.0
   Vs
1,000ppm

1,500ppm           52.73 [+ or -] 0.35    15.31 [+ or -] 0.25
                          n=14                   n=13

Control     P<             NS                    0.012
   Vs
1,500ppm

2,000ppm           53.18 [+ or -] 0.23    17.52 [+ or -] 0.1
                          n=19                   n=19

Control     P<             NS                     0.0
   Vs
2,000ppm

2,500ppm           53.42 [+ or -] 0.37    17.17 [+ or -] 0.21
                          n=18                   n=18

Control     P<             NS                    0.023
   Vs
2,500ppm

                          MCHC
                         (g/dl)

 Control           30.38 [+ or -] 0.45
                          n=28

1,000ppm           27.57 [+ or -] 0.24
                          n=10

Control     P<             0.0
   Vs
1,000ppm

1,500ppm           28.12 [+ or -] 0.49
                           n=9

Control     P<            0.002
   Vs
1,500ppm

2,000ppm           32.89 [+ or -] 0.1
                          n=19

Control     P<             0.0
   Vs
2,000ppm

2,500ppm           32.11 [+ or -] 0.24
                          n=18

Control     P<            0.002
   Vs
2,500ppm

Avearge [+ or -] SE; Significance (P) <0.05

* End of the experimental period
Hct = hematocrit

RDW = red distribution width

MCV = mean corpuscular volume

MCH = mean corpuscular hemoglobin

MCHC = mean corpuscular hemoglobin concentration