Chemoprotective effects of kolaviron on ethylene glycol monoethyl ether-induced pituitary-thyroid axis toxicity in male rats.

Endocrine disrupting chemicals cause reproductive dysfunction by interacting with intricate regulation and cellular processes involve in spermatogenesis. This study investigated the probable mechanism of action of ethylene glycol monoethyl ether (EGEE) as an antiandrogenic compound as well as the effects of kolaviron upon co-administration with EGEE in rats. Adult male rats were exposed to EGEE (200 mg kg(-1) bw) separately or in combination with either kolaviron [100 (KV1) and 200 (KV2) mg kg(-1) bw] or vitamin E (50 mg kg(-1) bw) for 14 days. Western blot analysis revealed that the administration of EGEE adversely affected steroidogenesis in experimental rats by decreasing the expression of steroid acute regulatory (StAR) protein and androgen-binding protein (ABP). EGEE significantly decreased the activities of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) but markedly increased sialic acid concentration in rat testes. EGEE-treated rats showed significant decreases in plasma levels of luteinising hormone (31%), testosterone (57.1%), prolactin (80.9%), triiodothyronine (65.3%) and thyroxine (41.4%), whereas follicle-stimulating hormone was significantly elevated by 76.9% compared to the control. However, co-administration of kolaviron or vitamin E significantly reversed the EGEE-induced steroidogenic dysfunction in rats. This study suggests that kolaviron may prove promising as a chemoprotective agent against endocrine pathology resulting from EGEE exposure.

Inhibition of metabolism of diethylene glycol prevents target organ toxicity in rats.

Diethylene glycol (DEG) is an industrial chemical, the misuse of which has led to numerous epidemic poisonings worldwide. The mechanism of its toxicity has not been defined as to the precise relationship between the metabolism of DEG and target organ toxicity. The purpose of this study was to investigate the mechanism for the acute toxicity of DEG, and the effect of the alcohol dehydrogenase inhibitor 4-methylpyrazole (fomepizole), by determining the relationship between accumulation of DEG or its metabolites and the resulting kidney and liver toxicity. Rats were treated by oral gavage with water, 2 g/kg DEG (low dose), 10 g/kg DEG (high dose), or 10 g/kg DEG + fomepizole, and blood and urine were collected over 48 h. Rats treated with high-dose DEG had metabolic acidosis, increased BUN and creatinine, and marked kidney necrosis, noted by histopathology. A minor degree of liver damage was noted at the high dose. After low and high doses of DEG, 2-hydroxyethoxyacetic acid (HEAA) was the primary metabolite in the urine, with only minor amounts of urinary diglycolic acid (DGA). Small amounts of ethylene glycol (EG), but not oxalate or glycolate, were observed in the urine. Treatment with fomepizole blocked the formation of HEAA and DGA and the development of metabolic acidosis and the kidney and liver toxicity. These results indicate that the mechanism for the target organ toxicity results from metabolites of DEG, and not DEG itself nor formation of EG from DEG, and that fomepizole may be a useful antidote for treating DEG poisoning.

Kupffer cells participate in 2-butoxyethanol-induced liver hemangiosarcomas.

2-Butoxyethanol increases hemangiosarcomas selectively in male mouse liver after chronic inhalation through mechanisms that have not fully been elucidated. Hemolysis, a primary toxic effect associated with 2-butoxyethanol exposure in rodents, increased hemosiderin (iron) deposition in Kupffer cells in the liver. These findings, along with the induction of hepatic neoplastic lesions, led to our hypothesis that the induction hemangiosarcomas by 2-butoxyethanol is due to the activation of Kupffer cells, subsequent to hemolysis, that results in the induction of DNA synthesis in target cells (endothelial cells); allowing for the selective proliferation of preneoplastic target cells and/or the promotion of new initiated cells. The present studies were conducted to determine whether Kupffer cells contributed to 2-butoxyethanol-induced endothelial DNA synthesis in the liver, thereby determining whether a linkage exists between these events. Male B6C3F1 mice were treated with 450 and 900 mg/kg 2-butoxyethanol (via daily gavage; 5x/week) for 7 days in the presence or absence of Kupffer cell depletion (via clodronate-encapsulated liposomes). 2-Butoxyethanol (450 and 900 mg/kg/day) increased the number of F4/80 stained cells (Kupffer cells) compared to controls (approximately 1.3- and approximately 1.6-fold over control, respectively). Clodronate liposome treatment reduced the number of Kupffer cells by >90%, as assessed by F4/80 immunohistochemistry. Increased hemolysis, measured by increases in relative spleen weights and decreased hematocrit was confirmed in 2-butoxyethanol treated mice. The percentage of iron-stained endothelial cells increased by approximately 11-fold over control, and endothelial cell DNA synthesis increased approximately 1.7-fold over control in 2-butoxyethanol exposed mice. Importantly, Kupffer cell depletion reduced 2-butoxyethanol-induced iron staining and hepatic endothelial cell DNA synthesis. These studies provide evidence supporting the hypothesis that the Kupffer cell modulates 2-butoxyethanol-induced endothelial cell DNA synthesis, and therefore may contribute to hemangiosarcoma induction by 2-butoxyethanol.

Suppression of the contact hypersensitivity response following topical exposure to 2-butoxyethanol in female BALB/c mice.

The effects of route of exposure, time of exposure and metabolism of 2-butoxyethanol (BE) on the contact hypersensitivity response (CHR) were evaluated in female BALB/c mice. Mice were either orally exposed to 50, 150 or 400 mg BE/kg or topically exposed to 0.25, 1.0, 4.0 or 16.0 mg BE on the ear and the oxazolone (OXA)-induced CHR evaluated by measuring ear thickness before and after OXA challenge. While no modulation was observed following oral exposure to BE, topical exposure resulted in a significant decrease in the CHR. Application of 4.0 mg BE in 4:1 acetone and olive oil (AOO) vehicle at the time of sensitization, challenge or both, decreased the CHR by 18%, 18% and 22%, respectively. A time course study of the effects of topical exposure to 4.0 mg BE/ear during the challenge phase of the CHR revealed that BE must be applied at the time of OXA challenge to significantly reduce the ear swelling response. In order to determine if metabolism of topically applied BE was required for suppression of the CHR, butoxyacetic acid (BAA), the primary metabolite of BE, was applied to the ear immediately following OXA challenge. No topical dose of BAA (2.0,4.0 and 8.0 mg BAA/ear) administered in this study altered the CHR. Blocking the metabolism of BE by oral administration of 4-methylpyrazole (MP), further reduced OXA-induced ear swelling when compared to mice exposed to BE without MP treatment. Taken together, these studies indicated that suppression of the CHR in mice following topical exposure to this glycol ether was due to the activity of BE itself and was not dependent on metabolic activation of the compound. Further studies were undertaken to identify a potential mechanism of BE-induced reduction of the CHR. Epidermal cells from untreated BALB/c mice were isolated and exposed to BE in vitro (10(-12), 10(-10), 10(-8), 10(-6) and l0(-4) M BE). In vitro exposure to BE at these concentrations did not significantly affect expression of MHC class II surface protein or protein synthesis in epidermal Langerhans cells, failing to provide in vitro evidence that BE-associated suppression of the CHR is associated with a reduction in MHC class II expression.

Co-administration of toluene and xylene antagonized the testicular toxicity but not the hematopoietic toxicity caused by ethylene glycol monoethyl ether in Sprague-Dawley rats.

Occupational painters are exposed to ethylene glycol monoethyl ether (EGEE), a widely used emulsifying solvent known to cause testicular degeneration and bone marrow depression, together with toluene (TOL) and xylene (XYL) as a mixture. In the previous study (Chung et al., Tox. Lett. 104:143, 1999), testicular atrophy caused by EGEE (200 mg/kg) was shown to be antagonized by co-administration of TOL (250 mg/kg) and XYL (500 mg/kg). This study was conducted to provide histological support for the previously observed antagonistic protective effect of TOL + XYL on EGEE inducible testicular toxicity and to determine whether a similar antagonistic effect can be demonstrated against the EGEE derived hematopoietic toxicity. Compared to the extent of seminiferous tubule degeneration caused by EGEE (150 mg/kg, six times per week for 4 weeks), testes of rats given co-administration of TOL (250 mg/kg) + XYL (500 mg/kg) showed dramatically reduced tubular degeneration. Hyperplasia of Leydig cells in the interstitium was observed in both EGEE and EGEE + TOL + XYL-treated rats. Although a minimal dose of EGEE causing testicular atrophy was used, WBC and platelet counts were decreased significantly. In the TOL + XYL-treated control group, the WBC and platelet counts were not decreased. However, the bone marrow depression caused by EGEE was not reversed by the combined administration of TOL + XYL. In all experimental groups (EGEE alone, TOL + XYL, EGEE + TOL + XYL), plasma levels of creatinine and alkaline phosphatase were significantly decreased. In addition to the marked testicular atrophy, EGEE also decreased the weights of adrenal glands and epididymis. In conclusion, while the testicular degeneration caused by EGEE was antagonized by TOL + XYL, the EGEE derived hematopoietic suppression was not reversed.

Serine-enhanced restoration of 2-methoxyethanol-induced dysmorphogenesis in the rat embryo and near-term fetus.

Effects of serine on restorative growth were characterized by comparing embryo/fetal responses after maternal exposure to 2-methoxyethanol (ME) and ME + serine by gavage on gestation day (gd) 13, a day of heightened limb sensitivity. Paws (gd 20) and limb buds (gd 15) were examined after ME alone at 50, 100, and 250 mg/kg, and after ME (either 100 or 250 mg ME/kg) + serine (1734 mg serine/kg) administered within minutes (0 hr) to 24 hr after ME. Paw development was not altered after ME at 100 mg/kg, but was highly sensitive to 250 mg ME/kg with all fetuses and litters exhibiting defects (ectrodactyly, syndactyly, and short digit) in the preaxial region. In contrast, the limb bud displayed dose-related incidences of abnormalities after maternal treatment with the low and high levels of ME. The condensing (precartilaginous, pentadactyl pattern) and noncondensing (undifferentiated mesenchymal cells) regions exhibited changes in their size, number, and location. Serine administration after 250 mg ME/kg was effective in reducing the occurrence of paw dysmorphogenesis with its protection potency inversely related to its delay of administration (i.e., 0% paw defect incidence after 0-hr delay, 25% after 4-hr delay, 41-45% after 8- and 12-hr delays, and 76% after 24-hr delay). The occurrences of limb bud pattern disturbances produced by ME were also markedly decreased by serine cotreatment. Higher incidences of embryonic defects versus those of fetal defects demonstrate that restorative growth followed ME exposure. Serine attenuation of ME teratogenicity appears to emanate from enhanced restorative growth so that tissue damage, which otherwise would be expressed as a defect at parturition, is repaired and replaced to resume development of the limb toward its normal structure.

Absence of high anion gap metabolic acidosis in severe ethylene glycol poisoning: a potential effect of simultaneous lithium carbonate ingestion.

Ethylene glycol poisoning is accompanied by a high anion gap metabolic acidosis. We describe a case of severe ethylene glycol poisoning in which severe acidosis was not present. This appears to be due to a beneficial effect exerted by simultaneous ingestion of 80 lithium carbonate tablets, each containing approximately 4 mEq carbonate, with a potential bicarbonate load of 320 mEq. We postulate a protective effect of lithium carbonate due to the bicarbonate generated by this substance.

Methoxyacetaldehyde, an intermediate metabolite of 2-methoxyethanol, is immunosuppressive in the rat.

2-Methoxyethanol (ME) is metabolized to 2-methoxyacetic acid (MAA) via the intermediate metabolite methoxyacetaldehyde (MAAD). Both ME and MAA have been shown in this laboratory to be immunosuppressive in rats following oral dosing. In this study, the plaque-forming cell (PFC) response to trinitrophenyl-lipopolysaccharide (TNP-LPS) was used to determine if MAAD is immunosuppressive in rats. Rats pretreated with the aldehyde dehydrogenase inhibitors disulfiram (2 mmol/kg) or cyanamide (0.48 mmol/kg) followed by oral dosing with ME (2.64 mmol/kg) resulted in suppressed PFC responses equivalent to the suppressed responses of rats dosed with ME alone. Rats pretreated with disulfiram and then dosed with 2.64 mmol/kg 2-methoxyethyl acetate (MEA), also resulted in suppressed PFC responses similar to that of MEA alone. In contrast, coadministration of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1.2 mmol/kg) with ME or MEA blocked suppression of the PFC response following exposure to ME or MEA alone. Oral dosing with equimolar (2.64 mmol/kg) concentrations of ME, MAA, or MAAD resulted in equivalent suppression of the TNP-LPS PFC response. Rats exposed to either disulfiram or cyanamide and MAAD also resulted in suppression of the PFC response. These results indicate that metabolism of ME to either MAAD or MAA is required for immunosuppression, and that these two metabolites are equipotent immunosuppressants in the rat.

Inhibition of intercellular gap junctional communication by alkyl ethers and its modulation by cAMP.

The inhibition of intercellular gap junctional communication (IGJC) by alkyl ethers (ethylene glycol, monomethyl ether, polyethylene glycol 1,000 and polyethylene glycol 6000) was examined using V79 Chinese hamster cells in vitro. Ethylene glycol and monomethyl ether inhibited IGJC very strongly, whilst the other agents inhibited IGJC only insignificantly. When the cells were treated with the combination of two agents, ethylene glycol and monomethyl ether, a significant increase in the inhibition of IGJC occurred. This was probably the result of potentiation rather than an addition effect. The effect of ethylene glycol was antagonized by dibutyryl cyclic adenosine monophosphate (DbcAMP). This effect was most intensive when the cells were treated with both agents at the same time and, in other experimental combinations, the effect was lower but also significant. Caffeine did not influence IGJC either in combination with DbcAMP or by itself.

Attenuation of 2-methoxyethanol-induced testicular toxicity in the rat by simple physiological compounds.

2-Methoxyethanol (2-ME) is an industrial solvent which is toxic to both male and female reproductive systems of laboratory animals. Earlier data have demonstrated that the developmental toxicity of 2-ME can be attenuated by simple physiological compounds such as serine, acetate, sarcosine, glycine, and D-glucose. The present experiments were designed to evaluate the same compounds for their ability to ameliorate the testicular toxicity that occurs in rats after 2-ME exposure. The extent of testicular damage was assessed by quantitating daily sperm production (DSP) on Day 24 following a single dose of 2-ME (6.6 mmol/kg, 500 mg/kg). Serine completely eliminated 2-ME-induced decreases in DSP, while glucose was without effect. Acetate, sarcosine, and glycine were of similar efficacy resulting in DSP that was significantly greater than that observed in rats which received 2-ME alone. Histopathological studies revealed that 2-ME treatment resulted in stage-specific degeneration of late stage pachytene spermatocytes 24 hr after treatment. No apparent degenerative changes occurred after concurrent treatment with serine. Similarly, serine also prevented the decreased number of spermatids in the lumina of the seminiferous tubules on Day 24 after 2-ME exposure alone. All of the compounds utilized in this study are linked to oxidation pathways involving tetrahydrofolic acid as a catalyst for one-carbon moiety transfer into purine and pyrimidine bases which are necessary precursors for DNA and RNA synthesis. The ability of these compounds to attenuate the testicular toxicity of 2-ME may result from their ability to donate one-carbon units which can be used in purine base biosynthesis. Reduced availability of bases would be expected to affect late stage pachytene spermatocytes which are known to be undergoing rapid RNA synthesis.

The possible role of one-carbon moieties in 2-methoxyethanol and 2-methoxyacetic acid-induced developmental toxicity.

The ethylene glycol ether, 2-methoxyethanol (2-ME), is rapidly (less than 1 hr) oxidized to 2-methoxyacetic acid (2-MAA). Both agents are selectively embryotoxic and equipotent in causing digit malformations when given to CD-1 mice on gestation Day 11. Previous observations have shown that simple physiological compounds such as formate, acetate, glycine, and glucose ameliorate the embryotoxicity of 2-ME. A common link for all of the attenuating agents may be oxidation pathways involving tetrahydrofolic acid (THF) as a catalyst for one-carbon transfer into purine and pyrimidine bases. In the present study serine at 16.5 mmol/kg, which reacts directly with THF, was as effective as formate in almost completely eliminating digit malformations resulting from treatment with 2-ME. Unlike formate, serine was equally effective against 2-MAA-induced dysmorphogenesis and the attenuating efficacy remained unchanged when serine administration was delayed for up to 8 hr after 2-ME or 2-MAA exposure. The protective effect of sarcosine, which is an intermediate in a pathway leading from choline to glycine and a structural analog of 2-MAA, was also determined. Both concomitant (43, 16.5, or 3.3 mmol/kg) and delayed (16.5 mmol/kg at 6 hr) sarcosine administration resulted in significantly less 2-ME-induced paw dysmorphogenesis. In addition, acetate administration was delayed for increasing intervals after 2-ME to determine the time at which attenuation would no longer occur, and acetate was effective for as long as 12 hr after 2-ME. These results support our hypothesis that 2-MAA, which has a long biological half-life, may interfere with the availability of one-carbon units for incorporation into purine and pyrimidine bases. Alterations in availability of these precursors might be expected to affect DNA and/or RNA synthesis and thereby influence normal cellular proliferation and differentiation in the developing embryo.