Zhang, Ming MD; Wang, Yanrang MD; Wang, Qian MD; Yang, Deyi MD; Zhang, Jingshu MD; Wang, Fengshan MD; Gu, Qing MD
* Become familiar with previous evidence on the neurotoxic effects of ethylbenzene, particularly hearing loss.
* Summarize the new findings on hearing loss in ethylbenzene-exposed petrochemical workers.
* Discuss the results of neurobehavioral function tests and neurotransmitter measurements in ethylbenzene-exposed workers.
Ethylbenzene is produced by alkylating benzene with ethylene, and mainly used as an intermediate in the manufacture of styrene.1 Neurotoxic effects have been observed in several animals after acute-duration exposure to inhaled ethylbenzene, although there is considerable variability in species sensitivity.2 In general, central nervous system depression is associated with acute exposure to higher concentrations of ethylbenzene, whereas stimulation of the motor nervous system is associated with lower ethylbenzene concentrations,2 which was similarly described in another study.3 The most serious adverse neurologic effect associated with acute- and intermediate-duration inhalation exposure to ethylbenzene is hearing loss, characterized by deterioration in auditory thresholds and alterations of cochlear morphology.4–6
As it is known to all, the nervous system works mainly depending on the normal variation of neurobiochemistry including neurotransmitters and neuropeptides, which play important roles in the physiologic and pathologic processes of the human nervous system. Neurotransmitters including monoamine, amino acid, and choline neurotransmitters regulate neurobehavioral function subtly. In this study, we investigated the levels of serum monoamine neurotransmitters, amino acid neurotransmitters, and choline neurotransmitters, the neurobehavioral function, and hearing loss in petrochemical workers to explore effects of ethylbenzene on blood neurotransmitters and neurobehavioral functions, as well as hearing.
MATERIALS AND METHODS
Study Populations and Sample Collection
The subjects of a cross-sectional study were workers from two different petrochemical plants in China, where the workers are exposed to both ethylbenzene and noise in their daily shift. During the manufactured process, plenty of ethylbenzene was used. From these two plants, 246 and 307 male workers were selected and classified into two ethylbenzene-exposed groups: petrochemical group 1 and petrochemical group 2. Two reference groups used were 290 male workers from a power station exposed to noise and signed as a power station group and 327 control subjects chosen from office personnel in these plants, with frequency matched to the exposed groups by age (±5 years). The response rate of the eligible exposed workers and controls that we approached for recruitment was approximately 95% and 90%, respectively. After obtaining their written informed consent to participate in this study, at enrollment all the individuals were interviewed; a questionnaire was used to elicit their health status, educational level, occupational history, smoking, and alcohol consumption. The blood and urine samples were collected from these subjects. Blood was prepared for hematologic detection, and separated by centrifugation for biochemical analysis.
Ethylbenzene Exposure Analysis
The ethylbenzene of various working environments was analyzed on the Shimadzu GC-14C gas chromatograph using ethylbenzene internal standards.5 The column temperature was maintained at 70°C. The injector and the detector were maintained at 275°C and 300°C, respectively. Nitrogen (purity >99.999%) was used as the carrier gas at a flow rate of 1.0 mL/min. Each sample of 1 μL was injected, and gas chromatography peaks were identified on the basis of the retention time of individual authentic standards (±0.3%). The abbreviation ND, which stands for not detected, means that the detected level is lower than the limit of detection (LOD), and is expressed with half of the values of LOD to enter the analysis.
Audiologic Assessment and Definitions of Hearing Loss
Pure tone audiometry was performed for both ears at 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, and 8.0 kHz. All auditory tests were performed in a sound-attenuating booth by a trained technician. Audiometry was carried out using Swiss Midimate RT-150 Audiometer (Brüel & Kjaer Company, Nærum, Denmark) calibrated to ISO 389 (1985-E) for the measurement of air conduction. Threshold value was defined as the lowest signal intensity that was detected in the subject at least 50% of the time, with a minimum of three tries. Masking was performed if the subject had a threshold value that differed by 40 dB or more between both ears. Otoscopic examination of the external acoustic meatus and tympanic membrane was done to exclude any ear diseases. Hearing loss can be in the either low-frequency range (0.5 to 2.0 kHz) or high-frequency range (4.0 to 8.0 kHz). We took the mean threshold of 0.5, 1.0, and 2.0 kHz (PTA512) as low-frequency hearing status and the mean threshold of 4.0, 6.0, and 8.0 kHz (PTA468) as high-frequency hearing status. Hearing threshold more than 25 dB in either low frequency or high frequency was defined as hearing loss.
Noise exposure levels at the selected workplaces were assessed with a sound pressure audiometer (BK-2231, Brüel & Kjaer Company, Nærum, Denmark) at 10 AM, 3 PM, and 5 PM for three consecutive days, twice per year, according to the Chinese national criterion for noise in the workplace. To evaluate the actual noise exposure level of the worker, cumulative noise exposure was calculated, on the basis of the database of a 20-year noise exposure, according to monitoring data on A sound pressure level and employment time calculated as follows: Expc = Leq + 16.61 × log10(T/T0), dB(A), where Expc is the cumulative noise exposure level; Leq, the time-weighted average exposed sound pressure level A; T, the total adjusted time worked (in years); and T0, year 1.
Test of Neurobehavioral Function
Neurobehavioral function was determined by the Neurobehavioral Core Test Battery recommended by the World Health Organization, consisting of the profile of mood state, simple response time, digital span, agile degree of lifting and rotating, digital decode, visual memory, and numbers of dots. The seven parts were mutually supplementary and evaluated the neurobehavioral function from the aspect of emotional state, learning and memory, and the degree of hand-eye coordination.
The levels of alanine transaminase, aspartate aminotransferase, total protein, albumin, alkaline phosphatase, and total bilirubin in serum were detected using commercial test kits (Jiancheng Bioengineering Ltd, Nanjing, China).
Red blood cells, white blood cells, hemoglobin and platelets were detected using commercial test kits (Jiancheng Bioengineering Ltd, Nanjing, China).
Dopamine (DA) Assay
Two hundred milliliters of 5% (v/v) perchloric acid was added into the same volume of serum, and then mixed sufficiently. After 20 minutes at a normal room temperature (∼23 °C), the samples were centrifuged for 20 minutes at 12,000 g/min at 4°C. The supernatants were centrifuged again and filtered through a microporous membrane with 0.45 μm pore diameter. Finally, the DA was analyzed from the supernatant by high pressure/performance liquid chromatography assay on the basis of this method.7
Gamma-Aminobutyric Acid (GABA) Assay
Four hundred milliliters of 2 mol/L potassium carbonate solution was added into 200 μL serum, and then 1400 μL 0.1 mol/L kalium carbonicum buffers with 50% methanol was added. The reaction system was mixed thoroughly and centrifuged at 14,000 g/min for 20 minutes at 4°C. The supernatants were centrifuged again at 14,000 g/min for 20 minutes. Then, 20 μL o-phthalaldehyde derivation reagents was added to the 40 μL supernatants, which had been filtered through a microporous membrane of 0.45 μm pore diameter, mixed, and then reacted accurately for 2 minutes at ambient temperature. The final 20 μL reaction solution was taken and determined the content of GABA with high pressure/performance liquid chromatography on the basis of this method.8
Ach and Acetylcholinesterase (AchE) Assay
The red blood cells were separated and dissolved in distilled water, which were called the hemolysis liquids and stored at −80°C for AchE activity analysis. AchE activity was assayed by 5,5′-dithio-bis-(2-nitrobenzoic acid) methods with special kits purchased from Nanjing Jiancheng Bio-Tech Corp, China. Ach was detected by alkaline hydroxyamine chromometry.
All data were entered into a computerized database. Further analysis was carried out using the statistical analysis software SPSS package (SPSS Inc, Chicago, IL). Measurements of continuous data were analyzed by univariate analysis of variance and t-tests. Categorical data were computed by the Pearson's chi-square contingency tables. The prevalence of hearing loss 25 dB or more in the ethylbenzene-exposed and noise-only groups were calculated. Using the administrative workers as the reference, odds ratios (ORs) and 95% confidence intervals (CIs) of hearing loss 25 dB or more were estimated for the ethylbenzene-exposed and noise-only groups, with adjustment for age, smoking tobacco, drinking alcohol, educational level, and employment time. A two-sided test with P < 0.05 was considered statistically significant.
General Characteristics of Investigated Population
The general characteristics of workers are presented in Supplemental Table 1 (see the Supplementary Digital Content Table 1, http://links.lww.com/JOM/A129). No significant differences in age, employment time, educational level, cigarette smoking, and alcohol drinking were found between the two groups.
Air Ethylbenzene Concentration
The levels of ethylbenzene were 122.83 ± 22.86 mg/m3 and 134.64 ± 31.97 mg/m3 in petrochemical groups 1 and 2, respectively, higher than that of the control group (not detected). The levels of other volatile aromatic hydrocarbons (styrene, benzene, toluene, and xylene) in all working environment plots were under LOD. The LOD of gaseous ethylbenzene and styrene, benzene, toluene, and xylene were 1.3, 1.7, 0.6, 1.2, and 3.3 mg/m3, respectively.
Noise Levels and Hearing Loss Information of the Investigated Population
The average noise exposure levels were 82.7 and 83.5 dB(A) in the two petrochemical sites, similar to 84.3 dB(A) in the power station site. The average noise exposure level in the offices of the control group was 67.3 dB(A).
The prevalence of hearing loss 25 dB or more was much higher in petrochemical group 1 (78.4%) and petrochemical group 2 (80.1%) than that in the power station (56.9%) and control (5.2%) groups (P < 0.05) (Table 1). Compared with the control group, the age-adjusted ORs for hearing loss 25 dB or more in the two petrochemical groups were 107 (95% CI = 17.1 to 358) and 114 (95% CI = 34.2 to 343), nearly four times greater than that of 27.5 (95% CI = 6.4 to 63.2) in the power station group, respectively.
Meanwhile, the multivariate logistic regression analysis (Table 2) indicated greater ORs of 86.4 (95% CI = 28.4 to 452) and 124 (95% CI = 11.7 to 343) for hearing loss in workers exposed to noise in petrochemical groups after the control of age, cigarette smoking, and alcohol drinking.
Neurobehavioral Function Changes in the Investigated Population
The results of neurobehavioral function changes in the investigated population are shown in Table 3. Compared with the control group, scores of fatigue–inertia, digital span, simple reaction time in petrochemical group 1 and scores of confused–bewilderment, digital span, simple reaction time in petrochemical group 2 were significantly enhanced, whereas the scores of Santa Ana manual dexterity test, Benton visual retention, and target tracking in the two petrochemical groups were depressed, respectively (P < 0.05).
The population of all petrochemical groups were further graded into five subgroups according the working age, and compared among the five groups. Table 4 indicates that the scores of five emotional states (tension, depression, anger, fatigue, and bewilderment) and simple reaction time in “3 to 4,” “4 to 5,” and “>5” working age sections were significantly higher than those in “<2” working age sections, respectively (P < 0.05). Compared with “<2” working age sections, scores of digital span in “4 to 5,” “>5” working age sections were remarkably depressed, as well as manual dexterity and digital symbol in “3 to 4,” “4 to 5,” and “>5” working age sections. The most of neurobehavioral function (tension, depression, anger, fatigue, bewilderment, simple reaction time, manual dexterity, and digital symbol) was significantly changed initially in the third year of working age. The significant difference of Benton visual retention, as well as target tracking, did not exist among all five groups (P > 0.05, respectively).
Changes of Neurotransmitters in the Investigated Population
Compared with the control group, the AchE activities (shown in Table 5) in the petrochemical groups were markedly decreased, respectively (P < 0.05). GABA, DA, and Ach showed no statistically significant difference between the control group and petrochemical groups (P > 0.05).
Correlation analysis indicated that the scores of digital span, simple reaction time, and Santa Ana manual dexterity positively correlated to AchE; the coefficient were 0.120, 0.170, and 0.153, respectively. No statistical correlation was found between other indexes and neurobehavioral function.
Changes of Biochemical and Hematologic Indexes in the Investigated Population
As shown in Supplemental Tables 1 and 2 (see the Supplementary Digital Content Tables 1 and 2, http://links.lww.com/JOM/A129), there was no significant difference between the control group and all exposed groups (P > 0.05).
Lines of evidences have indicated that ethylbenzene may induce neurotoxicity. In an archaic epidemiologic research, symptoms of dizziness accompanied by vertigo have been observed in humans acutely exposed to air concentrations of ethylbenzene ranging from 2000 to 5000 ppm for 6 minutes.9 Clinical signs of general central nervous system depression or increased motor activity have been observed in animals acutely exposed to inhaled ethylbenzene. Moderate activation in motor behavior was observed in rats after a 4-hour inhalation exposure to levels of ethylbenzene ranging from 400 to 1500 ppm, whereas narcotic effects were observed at higher ethylbenzene concentrations (2180 to 5000 ppm).2 Exposure of mice to ethylbenzene at 2000 ppm or more for 20 minutes produced changes in posture; decreased arousal and rearing; increased ease of handling; disturbances of gait, mobility, and righting reflex; decreased forelimb grip strength; increased landing foot splay; and impaired psychomotor coordination.10 No adverse effects were observed in the brain tissues of rats and mice exposed to chronic inhalation of ethylbenzene of up to 750 ppm for 2 years.11 In this study, we studied neurobehavioral function in ethylbenzene-exposed population. Neurobehavioral function representation significantly decreased in the exposed group, indicating the enhancement of fatigue, simple reaction time, digital span, decrease of vigor activity, Santa Ana manual dexterity test, Benton visual retention, and target tracking significantly compared with the control group. All these results hinted at the adverse alteration of short-term memory, quick hand movement, and hand-eye coordination among workers of the exposed group, involved with descending neurobehavioral function. Furthermore, most of the neurobehavioral function of them was significantly changed initially in the third year of working age. Ethylbenzene-exposed workers of 3-year working ages might be susceptible population of neurobehavioral function impairment, and they might be priority of protection. This indicated that adverse effects of ethylbenzene on neurobehavioral function were ahead of the alteration of clinical and biomedical indexes, which are shown in Supplemental Tables 2 and 3 (see the Supplementary Digital Content Tables 2 and 3, http://links.lww.com/JOM/A129).
Studies in laboratory animals identify hearing loss as the most sensitive end point for acute-duration inhalation exposure to ethylbenzene. A series of animal researches have demonstrated evidences of ototoxic effects of ethylbenzene exposure. Male rats exposed to ethylbenzene at 800 ppm, 8 hours per day for 5 days, indicated significant alteration in auditory thresholds 1 and 4 weeks after the exposure had ceased.4 Electrophysiologic research showed significant changes of auditory thresholds in rats exposed to ethylbenzene at 400 ppm or more.12 Another study indicated unchanged audition throughout the 13-week exposure period and the 8-week postexposure recovery period in rats exposed to ethylbenzene at 400 ppm or more.13 In humans, Barregård and Axelsson14 indicated synergistic ototoxic effects between organic solvent and noise, but the similar effect of unitary solvent component is not clear. An epidemiologic study of workers occupationally exposed to solvent mixtures that include ethylbenzene (mean ethylbenzene exposure level 1.8 ppm) for a mean of 13 years showed a 58% incidence of hearing loss compared with 36% in the control group.15 Hearing losses (indicated as enhanced hearing thresholds) were observed at all frequencies and seemed to range from 3 to 8 dB. Nevertheless, ethylbenzene was only one of the several solvents, most of which were even present at mean concentrations 1.5 to 3.5 times higher than ethylbenzene. Thus, hearing loss of these workers cannot directly be ascribed to the role of ethylbenzene. Our study indicated that the prevalence of hearing loss was higher along with increased employment time in the ethylbenzene-exposed group, which might be ascribed to otology of ethylbenzene. Essentially, the exposed levels of other volatile aromatic hydrocarbons (styrene, benzene, toluene, and xylene) were not detected.
To our knowledge, this study is the first to describe the effects of simultaneous exposure to ethylbenzene and noise on hearing loss in humans. In this study, the average noise exposure levels were similar between the petrochemical and noise-only groups. Nevertheless, the risk of hearing loss at 25 dB or more was much greater in the petrochemical group than in the noise-only group. This indicates that the risk of hearing loss induced by ethylbenzene exposure may be greatly higher than that induced by noise only.
Some signal transmitters in the central nervous system are involved in the process of cerebral ischemia, analgesia, learning and memory, and other neurophysiologic and neuropsychologic diseases.16 AchE regulates signal transmission among neurons in the brain by decomposing Ach. So AchE activity directly indicates the functional status of the cholinergic nerve system and is regarded as the marker enzyme. In this study, AchE activity decreased significantly in ethylbenzene-exposed population compared with the controls, while DA, GABA, and Ach showed a decreased trend yet no significant change. The results indicate that AchE neurotransmitter is involved in the neurotoxicity of ethylbenzene, guiding us to detect the contents of neurotransmitters in human peripheral blood, which may be surrogate of neurotransmitters in the central nervous system.
The Occupational Safety and Health Administration (OSHA) set a Permissible Exposure Limit (PEL) on the basis of a time-weighted average of 100 ppm (≈435 mg/m3 at 1 atm and 25°C) in the workplace.17 The American Conference of Governmental Industrial Hygienists (ACGIH) also recommends a Threshold Limit Value (TLV-TWA) of 100 ppm (≈435 mg/m3) for occupational exposures.18 The recommended exposure limit for occupational exposures (TWA) set by the National Institute for Occupational Safety and Health (NIOSH) is also 100 ppm (≈435 mg/m3) for ethylbenzene on the basis of a 10-hour average workday and a 40-hour workweek.19 Although the levels of exposed ethylbenzene in petrochemical groups 1 and 2 of our study were lower than limits of OSHA, ACGIH, and NIOSH, hearing loss, neurobehavioral alteration, and AchE elevation still occurred. Further studies carried out in large population should focus on the exposure limit of ethylbenzene in workplace.
In conclusion, occupational exposure to ethylbenzene might be associated with an elevated hearing loss and neurobehavioral function alteration, as well as with imbalance of AchE. This study herein has provided preliminary but important data for further study resulting from ethylbenzene. In addition, effective intervention is essential to improve individual occupational protection of workers who are exposed to solvents containing ethylbenzene.
This work was supported by the grants from a Key Project of Tianjin Municipal Science and Technology Pillar Program (No. 11ZCGYSY02100) and Key Projects of Tianjin Municipal Natural Science and Technology Foundation (No. 07JCZDJC08500, No. 09JCZDJC20900), Scientific Funds of Tianjin Bureau of Public Health (No. 12KY15), and Tianjin Centers for Disease Control and Prevention (CDCKY0902). The authors thank the participants of the work.
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