Subacute ethanol consumption reverses p-xylene - Science Direct

Toxicology, 75 (1992) 159-167 Elsevier Scientific Publishers Ireland Ltd.

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Subacute ethanol consumption reverses p-xyleneinduced decreases in axonal transport S. Padilla a, D . L . Lyerly b and C . N . P o p e c aU.S. Environmental Protection Agency, Health Effects Research Laboratory, Neurotoxicology Division (MD-74B), Cellular and Molecular Toxicology Branch, Research Triangle Park, North Carolina 27711, bBurroughs Wellcome Co., 3030 Cornwallis Rd., Research Triangle Park, NC 27711 and CToxicology Program, School of Pharmacy, Northeast Louisiana University, Monroe, Louisiana 71209 (USA) (Received March 16th, 1992; accepted June 21st, 1992)

Summary Human exposure to organic solvents is often complicated by ethanol ingestion and the literature is replete with demonstrations of metabolic interactions between ethanol and organic solvents at a pharmacokinetic level. Because of the possible modulation of xylene toxicity by ethanol consumption, the present group of studies characterizes the effect of ethanol on the p-xylene-induced decrease in axonal transport in the rat optic system previously reported by our laboratory. Long-Evans, hooded, male rats were divided randomly into two groups: those receiving 10% ethanol in their drinking water and those receiving water only. These two groups were further subdivided into two groups which were either exposed by inhalation to 1600 ppm p-xylene for 6 b/day, 5 days/week for 8 exposure-days or were treated identically except that they were exposed to air while in the inhalation chambers. The ethanol-drinking rats were given ethanol 6 days prior to and on the days of the inhalation exposure. Immediately after removal from the inhalation chambers on the last exposure day, the animals were injected intraocularly with [35S]methionine and [3H]fucose to measure the synthesis and rapid axonal transport of proteins and glycoproteins, respectively, in the retinal ganglion cells. The animals were sacrificed 20 h later, and the amount of radioactivity in different areas of the retinal ganglion cells was determined by liquid scintillation counting. As in previous experiments, the xylene exposure group showed a significant reduction in axonal transport of proteins and glycoproteins, whereas the ethanol exposure alone produced no significant reductions in the transport of either proteins or glycoproteins. In the animals receiving both ethanol and xylene, however, the ethanol treatment prevented the decreased transport characteristic of the xylene only animals, i.e. in all areas of the optic projections the level of transport were similar to the level present in the control groups. These data suggest that the xylene-induced reduction in rapid axonal transport was reversed (or prevented) by subacute ethanol consumption.

Key words." p-Xylene; Ethanol; Optic system; Axonal transport; Proteins; Glycoproteins; Rat

Introduction Organic solvents, as a class, have been implicated as neurotoxic agents in humans and laboratory animals. Xylene (dimethylbenzene), an aromatic hydrocarbon which Correspondence to: S. Padilla, U.S. Environmental Protection Agency, Health Effects Research Laboratory, Neurotoxicology Division (MD-74B), Cellular and Molecular Toxicology Branch, Research Triangle Park, North Carolina 27711, USA. 0300-483X/92/$05.00 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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is produced at an annual rate of billions of pounds, is used extensively as degreaser and cleaning agent, in aviation fuel, in the manufacture of plastics and synthetic fabrics [1] and as an 'inert' ingredient in some pesticide formulations. Chronic exposure to solvent mixtures containing xylene has been associated with the development of 'organic mental syndrome' in humans, a condition characterized by loss of memory, changes in personality, intellectual deterioration and possible peripheral neuropathy [2-4]. Ethanol ingestion is a factor frequently confounding human exposure to solvents. The metabolic interaction between xylene and ethanol has been documented but with conflicting results. Acute administration of ethanol increases the body burden ofxylene [5,6] and exacerbates the acute effects of xylene in humans [7-9]. Long-term (i.e. subchronic) ethanol intake, however, appears to lessen the body burden of inhaled xylene in rats [10,11]. Earlier studies from our laboratory have shown that inhalation exposure to 1600 ppm p-xylene for 6 h/day, 5 days/week for 8 exposure days produces a persistent, 30-50% decrease in rapid axonal transport of proteins and glycoproteins in the rat retinofugal projections [12]. This decrease may threaten the functional and structural integrity of the neuron as perturbations in axonal transport are often associated with neuronal degeneration [13-18]. This study was designed to assess the interaction between subacute ingestion of moderate levels of ethanol and the p-xyleneinduced decreases in protein and glycoprotein synthesis and axonal transport in the rat optic system. Materials and methods Animals and exposure parameters

Long-Evans male rats, 60-70 days old, 300-350 g (CrI:(LE)BR, Charles River Laboratories, Raleigh, NC) were housed in pairs on heat-treated pine bedding. The animals were placed individually into stainless steel mesh cages and exposed to pxylene (99% purity, Fisher Scientific, Raleigh, NC) in 11.4 cubic foot Rochester-type stainless steel chambers. The design, operation and monitoring of the chambers is described in detail elsewhere [12]. Animals were placed into xylene-free chambers and within 10 min the p-xylene concentration was within 10% of the target level and maintained at that level (4-10%) for the duration of the exposure period. The chambers were maintained at 68-70°F and 50-70% relative humidity throughout the exposure. The air control group was handled in exactly the same manner as the p-xylene exposed group except they were exposed to air instead ofp-xylene at the same time in a different chamber. Specifically p-xylene exposures were performed using either 0 or 1600 (± 160)ppm p-xylene for 6 h/day for 1.5 weeks (8 exposure days, 5 days/week). Animals were given either 0% or 10%(v/v) ethanol in their drinking water ad lib as their only liquid 6 days prior to commencing the inhalation exposure. While the animals were in the inhalation chambers (6 h/day) they did not have access to either food or liquid; however, each day after the animals were removed from the chambers (and on weekends), they had free access to both food (Purina Rat Chow) and either water or 10% ethanol. The entire experiment was conducted independently three times with n = 6-8 per

161 group per experiment. The results were qualitatively the same for each experiment and thus all data were pooled for analysis.

Measurement of axonal transport Immediately after the last exposure in the inhalation chambers (within 1 h), the animals were briefly anaesthetized with ether and the left eye of each animal was injected with 10/~Ci [3H]fucose (L-[6-3H]fucose, 16.8 Ci/mmol, NEN Dupont, Boston, MA) and 5t~Ci [35S]methionine (L-[aSS]methionine, 1097 Ci/mmol, NEN Dupont, Boston, MA) in 1.0 ~1 saline using a 10-#1 Hamilton glass syringe fitted with a 28-gauge needle (1-inch-long, point #4, specially manufactured by Hamilton Company Reno, NV). These intraocular injections are a relatively non-invasive and mild procedure. There is no bleeding, swelling, or irritation of the eye. The deep rectal temperature of each animal was measured using a Yellow Springs thermometer (Yellow Springs Instrument, Yellow Springs, OH) and body weights were recorded. The animals were decapitated 20 h after the isotope injection. The right and left retinas, optic nerves (a segment 5 mm proximal to the optic chiasm), optic tracts, lateral geniculate bodies and superior colliculi were removed and placed in 1.0 ml of ice-cold 10% trichloroacetic acid/0.5% phosphotungstic acid (TCA/PTA) to precipitate glycoproteins and proteins. These precipitates were washed, digested in 0.5 ml Protosol ® (NEN Dupont, Boston, MA), solubilized in 5.0 ml of scintillation cocktail [19] and counted in a Beckman LS 7000 scintillation counter. The maximum efficiency of the [3H]channel was 32% and the [35S]channel was 70%. All counts/min were corrected for quenching according to an external quench standard and converted to dpm. Levels of radioactivity in the structures labeled by axonal transport (left optic nerve and right optic tract, lateral geniculate and superior colliculus) were corrected for any systemic background labeling arising from precursor which leaked from the injected eye by subtraction of levels of radioactivity in the corresponding contralateral structures. Statistical procedures The amount of [3H] or [35S] radiolabel in each tissue piece was combined across the three independent experiments and the data compared using a three-way mixed analysis of variance (ANOVA). p-Xylene and ethanol were the between subjects factors (n = 19 to 21/group) while tissue piece was a repeated measure (General Linear Model Procedure; [20]). Results

The ethanol + xylene group was the only group which appeared overtly affected by the dosing regimen as their mean increase in body weight during the experiment (4.3% of the initial weight) was significantly less than the other three groups (12.1% in the water + air controls, 20.5% in the ethanol + air group and 12.1% in the water + xylene group; the weight gain in these three groups was not significantly different from one another). Ethanol administration alone for 16 days had no effect on the synthesis or rapid

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163 transport of radiolabeled proteins or glycoproteins in the rat retinofugal projections (Figs. 1 and 2). p-Xylene's effect on axonal transport of proteins (Fig. 1) or glycoproteins (Fig. 2) was definitely dependent upon the presence or absence of ethanol in the drinking water, p-Xylene inhalation produced a substantial decrease in transport of both the [35S]methionine labeled proteins (Fig. 1) and [3H]labeled glycoproteins (Fig. 2) to the axons of the retinofugal projections. There was a 50.3-57.8% decrease in the [35S]labeling of all areas of the retinal ganglion cell projections and a 43-58% decrease in the [3H]labeling. In contrast, the p-xylene-exposed animals receiving 10% ethanol in their drinking water showed a smaller reduction in axonal transport than did the animals receiving p-xylene alone. In fact, the amount of axonal transport in the ethanol + xylene group was not significantly different from the ethanol control group (i.e. those animals receiving ethanol but not p-xylene). The pattern of retinal labeling (see inserts in Figs. 1 and 2) was basically similar to that seen with the transport profiles, i.e. the only significant depression in labeling was noted in the animals exposed only to p-xylene and this depression in retinal metabolism was reversed by subacute consumption of 10% ethanol. If the data were 'normalized' by dividing the dpm present in each projection area piece by the dpm present in its respective retina, there were no significant differences between any of the treatment groups (data not shown). Discussion The present data confirm, as has been previously reported [12], that intermittent inhalation of p-xylene (1600 ppm, 6 h/day, 5 days/week, for 1.5 weeks) causes an extensive reduction in the amount of proteins and glycoproteins rapidly transported to the retinal projection sites following intravitreal injection of radiolabeled precursors. Also, as in that previous study, the decreased labeling of the retinal axons was accompanied by decreased labeling of the retina itself (insets in Figs. 1 and 2). Therefore, a likely explanation for the decreased axonal transport would be decreased synthesis of proteins and glycoproteins in the retina; however our previous experiments indicate that the rate of precursor incorporation was not changed by p-xylene exposure [12] (see Fig. 3 of Ref. 12). Additional implications of the decreased retinal labeling were considered in detail in the discussion section of that paper [12]. The results of this group of experiments indicate that animals maintained on 10% ethanol as a drinking liquid show less p-xylene-induced neurotoxicity than animals receiving no ethanol supplement. Ethanol administration may interact with xylene disposition (for a recent review on ethanol interactions see Ref. 21). Previous investigators have found that acute ethanol administration exacerbates the acute Fig. 1. [35S]methioninelabeling of the proteins in the projectionsof the retinal ganglion cells 20 h after intraocular injectionof methionine precursor. The retinal labeling is also shown (inset). Animals drank either 10% ethanol or water 6 days before and during the xyleneexposure (6 h/day, 5 days/week,for 8 days, 1600ppm p-xylene).Axonal transport was assessed during the 20 h followingthe last exposureday. Fig. 2. [3H]fucoselabeling of the glycoproteinsin the projectionsof the retinal ganglion cells 20 h after intraocular injection of fucoseprecursor. The retinal labeling is also shown (inset). Animals drank either 10% ethanol or water 6 days before and during the xyleneexposure (6 h/day, 5 days/week, for 8 days. 1600 ppm p-xylene). Axonal transport was assessed during the 20 h followingthe last exposure day.

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effects of xylene in humans [7-9], increases the blood concentration of xylene in rats [6] or humans [9] and decreases metabolic clearance of xylene in humans [5]; conversely, subacute administration of ethanol tends to reduce the body burden of xylene [10,11] after