* Environmental Health Science Bureau, Health Canada, Ottawa, Ontario, Canada and
College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801
Received July 16, 2003; accepted September 13, 2003
ABSTRACT
Pentyl ether (PE) and two newly synthesized polyoxy ethers, 1,4-diethoxybutane (DEB) and 1,6-dimethoxyhexane (DMH), have been proposed as candidate diesel fuel additives. To characterize and compare their toxicity and to provide information for risk assessment, a 4-week oral study was conducted on these compounds. Male Sprague-Dawley rats (288 ± 20 g) were divided into groups of seven animals each, and were administered by gavage low (2 mg/kg body weight), medium (20 mg/kg body weight), or high (200 mg/kg body weight) doses of PE, DEB, or DMH, respectively, 5 days/week for 4 weeks. Animals in the control group received the vehicle (corn oil, 1 ml/100 g body weight) only. At the end of the exposure period, relative testis and thymus weights were reduced by 30 and 46%, respectively, in animals treated with the high dose of DMH. Significant reductions in serum lactate dehydrogenase (LDH), serum uric acid, and blood platelet counts were also observed in the high dose of DMH. Serum corticosterone was significantly depressed in the high doses of PE and DEB and in the low dose of DMH. Serum thiobarbituric acidreactive substances (TBARS) were decreased (p < 0.05) in all DMH treatment groups and in the medium and high dose PE and DEB groups, while liver TBARS were unaffected by treatment. In the liver, increased glutathione (GSH) level and glutathione-S-transferases activity were detected in the high dose DMH group. Urinary ascorbic acid levels were markedly increased in animals receiving the high doses of PE, DEB, and DMH. Urinary formic acid was increased by 13 times in the high dose PE and DEB groups. Testes of all animals receiving the high dose of DMH showed a moderate to marked degree of degeneration of the seminiferous tubules, including a mild degree of vacuolation. At the same time, the epididymis of these animals had substantially reduced sperm density with prominent presence of spermatid giant cells. Mild histological changes were seen in the liver at all dose levels for all three chemicals. Thyroid effects were also observed in the high dose PE and DEB groups and in the medium and high dose DMH groups. It was concluded that DMH is the most toxic of the three ethers tested, with testicular, epidiymal, and thymic effects being the most prominent at 200 mg/kg. Other significant changes included depressed platelet counts and serum biochemical changes. Increased production of formic acid, an ocular toxin, from PE and DEB treatments may also be of toxicological concern.
Key Words: diesel fuel additives; aliphatic ethers; testicular effects; epididymal effects; thymic effects; formic acid.
Cetane, also known as n-hexadecane (C16H34), is a colorless, oily hydrocarbon and a reference fuel used to determine the ignition quality of diesel fuels. Cetane enhancers are important diesel fuel additives. Their presence in diesel fuels improves combustion quality and reduces hydrocarbon and carbon monoxide emissions in the exhaust (Heaton et al., 1993). Combustion quality is improved when ignition delay is shorter and cetane number is higher. Cetane number is a measure of the ignition quality of a diesel fuel that represents the percentage by volume of cetane in a mixture of
-methylnapthalene that gives the same ignition delay as the fuel being tested. Because of their high cetane number and miscibility, many aliphatic ethers, including pentyl ether (PE) and polyoxyethers 1,4-diethoxybutane (DEB) and 1,6-dimethoxyhexane (DMH), have been considered as candidate diesel fuel additives.
In the development of new fuel additives, it is important that the toxicity of these ethers be investigated and their potential health risks assessed at an early stage. Recently, the acute toxicity of a candidate additive, dimethoxymethane (methylal), was studied in rats, and its stability and toxicity were characterized (Poon et al., 2000). A review of the literature reveals that toxicity data is minimal for PE and nonexistent for DEB and DMH. The intravenous LD50 for PE was reported to be 164 mg/kg (Di Paolo, 1978
) in mouse. In this report, we studied and compared the systemic toxicity and histopathological effects of PE, DEB, and DMH on male rats following a short-term exposure. Because at low doses the effects may be subclinical in nature, sensitive biomarkers such as those for enzyme activities, hepatic effects (urinary ascorbic acid), and oxidative stress (TBARS, GSH) were also studied. In the studies of methyl tertiary butyl ether (MTBE), an ether oxygenate previously added to some gasoline, the formation of formaldehyde and its potential effects received much attention (Casanova and Heck, 1997
; Hutcheon et al., 1996
; Williams-Hill et al., 1999
). Therefore, the measurement of urinary formic acid, an end metabolite of formaldehyde, was included in the present study. Because the test compounds are moderately volatile (boiling points of 163188°C), it is expected some of the ingested compounds will be exhaled. Accordingly, biochemical markers of pulmonary effects were part of this study. An oral route of administration was chosen to emulate the exposure scenario of intake through potential contamination of drinking water (Stern and Tardiff, 1997
) and accidental ingestion of fuels (Litovitz et al., 1999
).
MATERIALS AND METHODS
Chemicals.
Pentyl ether (PE) was purchased from Sigma Aldrich (Oakville, Ontario, Canada); 1,6-dimethoxyhexane (DMH) and 1,4-diethoxybutane (DEB) were synthesized by Wychem Ltd (Suffolk, England). The purity of these three compounds was >99% as measured by gas chromatography. All other chemicals were of reagent grades and obtained from Fisher Scientific (Ottawa, Ontario, Canada) or Sigma Chemical Co. (St. Louis, MO).
Animal treatment.
Seven week old male Sprague-Dawley rats (Charles River Laboratories, St. Constant, Quebec, Canada) were divided into 10 groups of 7 animals each. They were housed in individual Health Guard® cages (Research Equipment Co., Bryant, TX) and were given food and water ad libitum. After 1 week of acclimatization, the groups were administered, by gavage, each of the test substances (PE, DEB, and DMH) in corn oil at one of the following doses: 2 mg/kg body weight (low), 20 mg/kg body weight (medium), or 200 mg/kg body weight (high). The oral administration was performed daily, 5 days/week, for 4 consecutive weeks. Control animals were administered corn oil (1.0 ml/100 g body weight) only. Body weights and food consumption were measured weekly. Cage-side observations were made weekly. The battery of observations consisted of the following: posture, clonic movement, gait score, piloerection, pitosis, lacrimation, salivation, vocalization, and ease of removal from home cage.
Overnight urine was collected from individual animals at the end of the fourth week. A portion of the urine was stored at -80°C for the determination of protein by a Lowry microprotein determination kit (Sigma Chemical Co.) and N-acetylglucosaminidase (NAGA) activity (Poon et al., 1995). The remaining urine was preserved in 0.4 N HCl/10 mM EDTA for the determination of ascorbic acid by an HPLC method (Poon et al., 1994
). All urine analyte concentrations were normalized against creatinine, which was measured enzymatically using a Roche creatinine assay kit (Roche Diagnostics, Quebec, Canada).
At termination, the animals were anesthetized with inhaled Isoflurane® (Baxter Corp., Toronto, Canada). Blood was withdrawn from the abdominal aorta for hematological analysis and serum clinical chemistry. The right lung was clamped and the left lung was instilled with saline (17.5 ml/kg body weight) according to the method of Hatch et al. (1986). The lavage fluid was centrifuged at 700 x g for 15 min and the cell-free bronchoalveolar lavage fluid (BALF) was stored at -70°C pending the analysis for protein and NAGA, using the methods described above for urine. The brain, heart, thymus, liver, kidneys, spleen, and testis were excised and weighed. A 2 g piece of liver was removed and homogenized in 3 ml of 0.05 M Tris/0.15% KCl buffer, pH 7.4. Part of the homogenate was centrifuged at 9000 x g for 20 min to obtain the S9 fraction. Both the homogenates and S9 fractions were stored at -80°C until use. The liver, kidneys, thymus, thyroid, and epididymis were preserved in phosphate-buffered formalin. Testes were fixed in Bouins solution, washed in 70% ethanol, and preserved in phosphate-buffered formalin. The fixed tissues were dehydrated with graded alcohol, cleared, and impregnated in paraffin. The paraffin blocks were sectioned to 5 µm thickness and stained with hematoxylin and eosin for microscopic examination.
Hematological and biochemical analysis.
A Technicon H1E hematology analyzer (Bayer, Toronto, Ontario, Canada) was used for the determination of the following hematological parameters: erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet count, white blood cell counts, and percentages of lymphocytes. A Technicon RA-XT analyzer was used to obtain the following serum chemistry parameters: aspartate aminotransferase activity, alkaline phosphatase activity, total protein, albumin, inorganic phosphate, urea nitrogen, creatinine, glucose, and cholesterol. The traditional lactate dehydrogenase (LDH) activity as well as the LDH-X activity were measured in serum using lactic acid and DL-hydroxycaproic acid, as the substrate, respectively (Blanco et al., 1976
). Serum testosterone was determined using a radioimmunoassay kit (ImmunChem, ICN Pharmaceutical Inc., Costa Mesa, CA), while serum corticosterone was measured using an HPLC procedure (Wong et al., 1994
).
Reduced glutathione (GSH) was measured in liver homogenates using a colorimetric kit method (Calbiochem, La Jolla, CA). Glutathione-S-transferase (GST) activity was measured in the liver S9 fractions (Habig et al., 1974). Thiobarbituric acidreactive substances (TBARS) in the liver homogenates and serum were determined by a fluorescence method (Yagi, 1982
).
Statistical analysis.
One-way ANOVA and Duncans multiple range test were used to identify groups that were significantly different from the control at the p < 0.05 level.
RESULTS
Food consumption and body weight gain of animals treated with PE, DEB, or DMH did not differ significantly from the control animals (Table 1). None of the treated animals exhibited clinical signs of toxicity. Animals receiving the high dose of DMH (200 mg/kg) had significantly smaller testes and thymus gland, with the average weights of these organs relative to body weight decreased by 30 and 46%, respectively, compared to the control group (p < 0.05; Table 1
). The relative weights of the brain, heart, liver, spleen, and kidneys were unaffected by treatment.
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Serum TBARS levels were decreased (p < 0.05) in all of the DMH treatment groups and in groups receiving the medium and high doses of PE and DEB (Table 3). No significant changes in TBARS were observed in the liver.
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Among the three aliphatic ethers studied, DMH at 200 mg/kg produced the most conspicuous adverse effects of testicular and thymic atrophy. The histopathological basis for the observed testicular atrophy is clearly related to degenerative changes occurring in the seminiferous tubules consisting of loss of epithelium, loss of developing spermatids, and reduced tubular diameter. The markedly reduced sperm density observed in the epididymides of these animals can be interpreted as a functional manifestation of the lesions in the seminiferous tubules resulting in a decline in sperm production (Ku et al., 1993). Although the reduction in epididymal sperm density was severe, a multigenerational study is required in the future to evaluate the impact of DMH on fertility (Chapin et al., 1997
). A limitation of the present study is that the staining technique precluded a stage-specific screening for affected cell types (Hess, 1990
). However, the prominent presence of spermatid giant cells in the epididymides provided supporting evidence that degenerative changes were occurring in the seminiferous tubules and affecting spermatogenesis (Holstein and Eckmann, 1986
; Morton et al., 1986
; Somkuti et al., 1991
). A wide variety of testicular toxicants induced the appearance of multinucleated spermatid giant cells (Anderson et al., 1992
; Dodd et al., 1994
; Working et al., 1985
), often accompanied by a reduction of sperm density in the epididymis (Fail et al., 1990
; Somkuti et al., 1991
).
The absence of LDH-X, a testicular isoyme released into the circulation by damaged testes (Reader et al., 1991), provided a biochemical indication that there were no acute testicular injuries. However, this does not preclude the possibility that testicular injuries had occurred at earlier time points. The lack of treatment-related changes in serum testosterone suggested that interstitial cells, the major site of testosterone release, were unaffected by all three ethers.
There are two industrial solvents with structures that bear some similarity to that of DEB and DMH: 1,2-dimethoxyethane and 1,2-diethoxyethane, also known as ethylene glycol dimethyl ether and ethylene glycol diethyl ether, respectively. Both compounds have been shown to be embryotoxic and developmental toxins (George et al., 1992; Hardin and Eisenmann, 1987
; Leonhardt et al., 1991
; Nagano et al., 1984
). Nagano et al. (1984)
demonstrated that mice treated with 1,2-dimethoxyethane at oral doses of 250 mg/kg or higher for 5 weeks had decreased testicular weight, atrophy of the seminiferous tubules with markedly reduced sperm density, and generally normal Sertoli and Leydig cells. In the present study, DMH-induced testicular atrophy was also accompanied by reduced sperm density in the epididymides. It is interesting to note that another structurally related compound, bis(2-methoxyethyl) ether (diglyme), also induced testicular atrophy, degeneration of spermatocytes, and spermatid giant cells following 20 daily doses at 684 mg/kg (Cheever et al., 1989
). 2-Methoxyacetic acid, which is known to produce testicular and thymic atrophy (Miller et al., 1982
; Moss et al., 1985
), has been proposed to be the metabolite responsible for the toxic effects observed in bis(2-methoxyethyl) ether and 1,2-dimethoxy ethane-treated animals (Daniel et al., 1991
; Hardin and Eisenmann, 1987
). Given the observed effects of DMH on the testis, epididymis, and thymus, it can be postulated that 2-methoxyacetic acid may be the predominant metabolite responsible. It can also be postulated that DEB was without effect at the present dose levels because one of its major metabolites, ethoxyacetic acid, is known to be less toxic than methoxyacetic acid (Foster et al., 1987
; Smialowicz et al., 1992
).
Miller and coworkers have shown that 2-methoxyacetic acid induced a reduction in bone marrow cellularity (Miller et al., 1982). They (Miller et al., 1984
) also demonstrated that 2-methoxyethanol (ethylene glycol monomethyl ether), which was metabolized to 2-methoxyacetic acid (Medinsky et al., 1990
), caused a decrease in platelet counts in rats and rabbits. It can be postulated that the DMH-associated depression in platelet counts may also be mediated by 2-methoxyacetic acid as a metabolite.
Rodents responded to a wide variety of hydrocarbon hepatotoxins by increasing the excretion of ascorbic acid (Burns et al., 1960; Poon et al., 1994
). Typically, organochlorine-type hepatotoxins stimulated ascorbic acid excretion at intake levels of 15 mg/kg/day (Chu et al., 1998
; Poon et al., 1999
). The fact that all three ethers stimulated ascorbic acid excretion at 200 mg/kg indicated that they were comparatively much less potent as a hepatotoxin. Indeed, considering as a whole the reversible nature of the histopathological changes, the absence of hepatomegaly, and the normal serum aspartate aminotransferase activity, it can be concluded that the hepatic effects were adaptive and reversible in nature.
Changes in the thyroid were reversible in nature; the minimal reduction in colloid density suggested that the functional aspects of the thyroid may not be adversely affected.
Although DMH induced frank thymic atrophy, architectural and cytologic abnormalities were not detected at the histological level. Thymic atrophy in rodents has been reported to be correlated with increased serum corticosterone (Cuff et al., 1996; Gorski et al., 1988
) or directly induced by the glucocorticoid analog dexamethasone (Lundberg, 1991
; Sun et al., 1992
). In the present study, serum corticosterone did not appear to be a cause of thymic atrophy because its level decreased rather than increased in the treated animals. Further studies focusing on the effects of DMH on the phenotypes and cellular responses of thymocytes will be needed to understand the mechanisms of toxicity.
Urinary protein and NAGA are sensitive biomarkers of injuries to proximal tubular cells. The absence of increases in these two parameters, together with a lack of treatment-related increases in kidney weights, indicated that the three aliphatic ethers are not likely to be nephrotoxic. Cytoplasmic inclusion of hyaline droplets in the proximal tubular cells is a change that is specific to the males of the rodent species (Halder et al., 1985; Short et al., 1989
). Therefore, the accentuation of this change and the associated cytoplasmic shedding, mainly in the high dose DMH group, may not have toxicological implications in humans.
Protein and NAGA levels in cell-free bronchoalveolar lavage fluids are also sensitive biomarkers of early injuries in the lung (Henderson et al., 1979; Poon et al., 1995
). The lack of significant increases of these biomarkers in the treated animals suggested that the aliphatic ethers were not pulmonary toxicants.
A biochemical effect unique to DMH is the increased liver GSH and GST activity. These increases suggested that there was an enhancement in the livers capacity to metabolize high doses of DMH via the mercapturic acid detoxification pathway.
None of the aliphatic ethers induced oxidative stress as judged by the absence of increased TBARS in the liver and serum. It is not clear at this point whether reduced serum TBARS signifies that the aliphatic ethers, DMH in particular, possess some antioxidant properties. The marked stimulation of urinary excretion of formic acid in of PE- and DEB-treated rats is a novel observation. Formic acid is an endogenous metabolite (Kavet and Nauss, 1990) found in the urine, normally at the range of 1.2 to 17.5 mg/g creatinine (Trieberg and Schaller, 1980
). The magnitude of increase in urinary formic acid in the high dose PE and DEB groups is comparable to that found in urine of rats following intraperitoneal injection of 100 mg/kg methanol (Kornbrust and Bus, 1982
). It is not known if the increase is related to a perturbation in one-carbon metabolism, increased lipid peroxidation, changes in renal excretion, or the metabolism of the aliphatic ethers themselves. Some small molecular weight hydrocarbons are known to be metabolized to formaldehyde through oxidative demethylation (Cederbaum and Cohen, 1980
; Kukielka and Cederbaum, 1995
). Formic acid was considered the proximal toxicant responsible for adverse effects associated with acute methanol poisoning, and humans and other primates are known to be more susceptible (Kavet and Nauss, 1990
; McMartin et al., 1980
). It remain to be seen whether acute exposure to aliphatic ethers in diesel fuels can raise the formic acid to levels that can be considered to be toxic in humans.
Returning to the primary focus of this study, i.e., the comparative toxicity of candidate diesel additives, DMH at 200 mg/kg clearly produced the most severe adverse effects. The most prominent of these are thymic and testicular atrophy, degeneration of seminiferous tubules, and reduced epididymal sperm density, and they may be related to metabolites such as 2-methoxyacetic acid. Other significant effects included decreased platelet counts and serum uric acid. All three ethers at 200 mg/kg produced mild and reversible histological changes in the liver and thyroid gland. The presence of high levels of formic acid, an ocular toxin, in animals dosed with PE and DEB may be of health concern during acute poisoning.
ACKNOWLEDGMENTS
The authors wish to thank A. Yagminas for his expert advice, and B. Nadeau, S. Masson, Y. Dirieh, and T. Odorizzi for their excellent technical assistance. This work was supported in part by the Federal Programme on Energy Resource and Development.
NOTES
1 To whom correspondence should be addressed at Health Canada, Environmental Health Center, locator No. 0803 B, Tunneys Pasture, Ottawa, Ontario, Canada K1A 0L2. Fax: (613) 957-8800. E-mail: raymond_poon{at}hc-sc.gc.ca.
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