Hematotoxicity induced by simultaneous exposure to noise and toluene in New Zealand white rabbits: Synergistic and antagonistic effects

Abstract

Exposure to numerous pollutants is prevalent in workplaces. Examination of combined exposure to different harmful physical factors and chemicals has offered new insights into toxicology in recent years. This study aimed to investigate the hematological alterations caused by exposure to noise and toluene. Twenty-four New Zealand white rabbits were exposed to 1000 ± 50 ppm toluene and/or 100 ± 5 dB noise for 14 consecutive days. Exposure to noise and toluene changed a number of parameters of white blood cells (WBC), red blood cells (RBC), and platelets on different days after the exposure. Simultaneous exposure to noise and toluene increased WBC, and exposure to noise and toluene alone decreased RBC. Exposure to noise and toluene alone increased basophile, monocyte, and neutrophil counts. The coefficient of variation of red blood cell distribution width (RDW-CV) and the standard deviation of red blood cell distribution width (RDW-SD) significantly increased after co-exposure to noise and toluene. Platelet levels increased in the noise-exposed and the co-exposed groups and decreased in the toluene-exposed group. Furthermore, co-exposure to noise and toluene induced dissimilar synergistic and antagonistic effects on the hematological indices. According to the results of this study, simultaneous exposure to toluene and noise can aggravate some hematotoxic effects compared to exposure to noise or toluene alone. The results also demonstrated the vital role of the modulatory mechanisms of the body in controlling the detrimental effects of stressors.

Keywords

Pollution industrial toxicology exposure noise toluene

Introduction

Exposure to noise is widely considered one of the most important factors inducing hematological changes (Alimohammadi and Danesh, 2014; Kavita Marita et al., 2012; Vazzana et al., 2017) and immunological reactions (Cui et al., 2016). Noise and toluene are two of the most prevalent physical and chemical stress factors (Basner et al., 2014; Abouee-Mehrizi et al., 2021).

Transportation and industrial activities are the main sources of noise pollution. In acute exposures, the range of noise intensity is usually 90 dB–110 dB (Abouee-Mehrizi et al., 2020a; Vlajkovic et al., 2004). In recent years, noise has been found to be a harmful biological (Basner et al., 2014), psychological (Park and Lee, 2017), and immunological risk factor (Zheng and Ariizumi, 2007; Pascuan et al., 2014; Abouee-Mehrizi et al., 2020a).

Toluene is used in industries as an industrial chemical and a solvent for numerous applications (Abdi Khanghah et al., 2017; Abouee-Mehrizi et al., 2022). Acute exposure to toluene mostly occurs in chemical industries above 200 ppm (Michael and Utidjian, 1974; Asenjo et al., 2011; Fishbein, 1985; Kobald et al., 2015). Vertigo, headache, anesthesia, respiratory distress, skin dryness, neurological disorders, and depression due to exposure to toluene have been widely examined (Hume and Ho, 2019).

Some studies on the hematological effects of noise showed that exposure to noise decreased red blood cell levels (Zymantiene et al., 2017; Axelsson and Dengerink, 1987; Qureshi et al., 2002). Moreover, exposure to some organic solvents such as toluene decreases hematocrit levels in petrochemical industries (Neghab et al., 2014). Furthermore, some studies on hepatotoxicity and nephrotoxicity of noise and toluene indicated a wide range of adverse effects on liver and kidney tissues, which can cause or aggravate hematological disorders (Abouee-Mehrizi et al., 2020b, 2021).

It has been shown that co-exposure to noise and toluene happens in many industries such as footwear, wood furniture, and petrochemical industries (Schäper et al., 2008; Chang et al., 2006; Sung et al., 2005). However, there is a lack of valid scientific studies into the effects of simultaneous exposure to noise and toluene on blood parameters in humans and even in laboratory animals. This study was conducted because of the dearth of studies presenting the hematological effects in rabbits after exposure to noise and toluene.

Materials and methods

Experimented animals

Twenty-four male New Zealand white rabbits (4 months old, weighing 2.83 ± 0.41 kg) were used. The animals were kept on a 12:12-h day/night cycle and specified temperature (20 ± 5°C) and humidity (50–70%) with free access to treated water and standard pellets (Thermo-hygrometer, Kimo Model: HD 100, E, Mumbai, Maharashtra 400,055, India). They were exposed 14 days after placement in the laboratory to accommodate the laboratory environment.

Experimental groups

The animals were separated into four groups, and six animals were randomly placed in each group. After assigning a number to each animal, simple random sampling was performed. Group 1 was only exposed to noise; Group 2 was only exposed to toluene; Group 3 was co-exposed to noise and toluene; and the control group (Group 4) was only exposed to clean air devoid of any toluene or noise stress (≤50 dBA). Exposure time was 8 h/day for 14 consecutive days for all groups. Humidity and temperature in the exposure chamber were constantly measured using a humidity dew point meter (Thermo Hygrometer, Kimo Model: HD 100, E, Mumbai, Maharashtra 400,055, India) through control outlets designed on the side walls and ceiling of the exposure chamber during exposure for all groups. The exposure chamber and environmental conditions such as relative humidity (50–70%), air flow rate (33 L/min), and temperature (20 ± 5°C) were maintained for all groups as long as possible for the period of exposure, and each group was placed separately in the exposure chamber until the end of the exposure period.

Exposure chamber features

The exposure chamber was designed based on previous studies (Gad, 2006; Cobo et al., 2009; Moreno et al., 2000; Abouee-Mehrizi et al., 2020b). The size of the exposure chamber was 50*60*90 cm and it was built with transparent polycarbonate sheets.

To mix clean air and toluene vapors before entering the exposure chamber, fresh air and toluene vapors were combined in a mixer chamber with the same material as the exposure chamber and with a size of 50×50×20 cm. The purity of the toluene generated in the chambers was confirmed using a gas chromatography-mass spectrometry device (Agilent 6890/5973 inert GC/MSD, Mike Szelewski Agilent Technologies, Inc, USA) in the form of pilot tests before the actual exposures.

Experiments

The following experiments were performed:

Noise exposure system

The white noise of 100 ± 2 dBA was generated using Audacity® 1.3.12 Beta and controlled online by Cool Edit Pro 2.1. A speaker, a laptop, and an amplifier (3030 W, Multi Tone) were used to generate and regulate the noise level. The noise source (speaker) was fixed on the ceiling of the exposure chamber (Figure 1).

The frequency and intensity of noise were checked every 30 min using a TES 1358 Sound Analyzer Real-Time device.

The clean air flow rate was 33 L/min, without any toluene vapors. The airflow rates were checked using two calibrated flow meters (Platon, Roxspur Measurement and Control Ltd, England) in the two air hoses: one hose with a high volume (to check 30 L/min) and the other hose with a low volume (to check 3 L/min).

Toluene exposure system

According to the results of the pilot experiments, at the beginning of the toluene generation, an impinger was filled with 100 cc of toluene liquid (Merck 1.08,323.2500 extra pure, 64,271 Darmstadt, Germany). The fresh air flow rate was 30 L/min and the toluene vapor flow rate was 3 L/minute, checked by two flow meters with the low volume for toluene vapors and high volume for fresh air (Platon, Roxspur Measurement and Control Ltd, UK). Consequently, the total flow rate was like that of the other groups (33 L/minute). Toluene concentration was regularly monitored through control outlets every 30 min during both pilot tests and real exposures using a phocheck (Ion Science Ltd, Cambs UK). Moreover, the concentration of toluene was monitored during the pilot tests using a low-flow sampling pump (SKC 222–3, USA) by active charcoal adsorbent testers and using a gas chromatography device (Agilent 7890A GC System).

For the pilot tests, due to the reduction of the toluene liquid in the impinger during the exposure, 20 ccs of toluene were poured into the impinger every 90 min, and this was recurrent until the end of the 8 hours of each exposure every day to eliminate the alternation of toluene concentration in the exposure chamber.

Blood collection

Blood samples were collected from a marginal vein of the ear of each animal into EDTA-containing tubes (K2 EDTA CBC, Kang Jian, China). The ears were warmed using warm cotton before each blood sampling. We chose five periods of blood collection to investigate the trend of variations in hematological parameters on different post-exposure days. Period 1 was immediately before exposure; Period 2 was immediately after exposure; Period 3 was 3 days after exposure; Period 4 was 7 days after exposure; and Period 5 was 14 days after exposure.

Assessment of hematological parameters

After blood sampling, 1 cc of all the collected samples was kept in the CBC blood tubes (containing anticoagulant (EDTA)) to measure hematological factors using a blood cell counter machine (H1-Technicon Instruments Corporation, Tarrytown, NY, USA). The measured blood parameters were red blood cells (RBC), mean corpuscular volume (MCV), mean platelet volume (MPV), mean corpuscular hemoglobin (MCH), hemoglobin (HGB), hematocrit (HCT), procalcitonin (PCT), platelet (PLT), platelet distribution width (PDW), coefficient of variation of red blood cell distribution width (RDW-CV), the standard deviation of red blood cell distribution width (RDW-SD), white blood cells (WBC), monocyte, and eosinophil.

Statistical analysis

SPSS (25.0.0.0, Inc., Chicago, IL, USA) was used for data analysis. To compare changes between different repeats in different groups, the generalized estimating equations (GEE) method was applied. The statistical significance level was 0.05.

Results

Erythrocytes

RBC significantly decreased in the noise-exposed group compared to the control group in Periods 2, 3, and 4. Moreover, RBC in the toluene-exposed group was significantly lower than in the control group in Periods 3, 4, and 5 (Figure 2(a)). HGB in the noise-exposed group in Period 3 was significantly lower than in the control group in Periods 3 and 4. Moreover, HGB in the toluene-exposed group was significantly lower than the control group in Periods 4 and 5. (Figure 2(b)).

HCT level in the noise-exposed group was significantly lower than in the control group in Periods 2, 3, and 4. Moreover, the HCT level in the toluene-exposed group was significantly reduced compared to the control group in Periods 3, 4, and 5. The HCT level in the co-exposed group was lower than in the control group in Periods 2, 3, and 4 (Figure 2(c)).

The MCH level in the noise-exposed group significantly decreased compared to the co-exposed group in Periods 4 and 5. Furthermore, the MCH in the noise-exposed group was significantly lower than the control group in Period 4 (Figure 2(d)).

The MCV level in the toluene-exposed group in Periods 2, 3, and 5 was significantly lower than the control group in Period 5. Moreover, MCV in toluene and co-exposed groups was significantly lower than in the control group in Period 2 (Figure 2(e)).

RDW-CV in the noise-exposed group was significantly higher than the co-exposed group in Period 2 and significantly higher than the control group in Period 5 (Figure 2(f)). RDW-SD in the noise-exposed group was significantly higher than the co-exposed group in Period 2 and significantly higher than the control group in Period 5. In addition, RDW-SD in the toluene-exposed group was significantly higher than in the control group in Periods 3 and 5 (Figure 2(g)).

Leukocytes

The WBC level in the co-exposed group was significantly higher than in the noise-exposed and toluene-exposed groups and significantly higher than the control group in Period 2 (Figure 3(a)). The lymphocyte level in the noise-exposed group was significantly lower than in the co-exposed group in Periods 2 and 3 and significantly lower than the control group in Period 4. Moreover, the lymphocyte level in the toluene-exposed group was significantly lower than in the co-exposed group in Periods 2 and 3 and significantly lower than the control group in Periods 2 and 4. Additionally, the lymphocyte level in the co-exposed group was significantly higher than the control group in Period 2 (Figure 3(b)).

The neutrophil level significantly increased in the noise-exposed group compared to the control group in Period 2. The neutrophil level in the toluene-exposed group significantly increased compared to the control group in Period 2. The neutrophil level also significantly increased in the co-exposed group compared to the control group in Period 2. Furthermore, the neutrophil level in the co-exposed group was significantly lower than the toluene-exposed and control groups in Period 3 and significantly lower than the noise-exposed group in Period 4 (Figure 3(c)).

The monocyte level in the noise-exposed group was significantly higher than the toluene-exposed and control groups in Period 3 and significantly higher than the control group in Periods 2, 4, and 5. The monocyte level significantly increased in the toluene-exposed group compared to the co-exposed group in Period 5. Moreover, the monocyte level in the toluene-exposed group was significantly higher than the control group in Period 5 (Figure 3(d)).

The level of eosinophil significantly decreased in the toluene-exposed group compared to the control group in Period 2. Furthermore, the eosinophil level in the co-exposed group was significantly lower than the control group in Periods 3 and 4 (Figure 3(e)).

The basophil level in the noise-exposed group was significantly higher than the co-exposed group in Period 4 and significantly higher than the control group in Periods 2 and 4. The basophil level in the toluene-exposed group was significantly higher than the control group in Periods 2 and 3. Furthermore, the basophil level in the co-exposed group was significantly lower than the noise and toluene-exposed groups in Period 2. The basophil level in the co-exposed group was significantly higher than in the toluene-exposed and control groups in Period 3. Moreover, the basophil level in the co-exposed group was significantly higher than in the toluene-exposed group in Period 5 (Figure 3(f)).

The PCT level in the noise-exposed group was significantly higher than the control group in Period 2. The PCT level significantly decreased in the toluene-exposed group compared to the noise-exposed and control groups in Period 2. Moreover, the PCT level in the co-exposed group was significantly lower than the control group in Periods 3 and 4 (Figure 4(a)).

Platelets

MPV in the noise-exposed group was significantly reduced compared to the control group in Period 3; also, MPV in the toluene-exposed group was significantly lower than the control group in Periods 2 and 3. MPV was significantly reduced in the co-exposed group compared to the control group in Periods 3 and 4 (Figure 4(b)).

PDW in the noise-exposed group was significantly lower than in the control group in Period 3. PDW in the co-exposed group was significantly lower than the control group in Period 3 and significantly higher than the noise-exposed group in Periods 3, 4, and 5 (Figure 4(c)).

The PLT level in the noise-exposed group significantly increased compared to the control group in Period 2. The PLT level had a significant decrease in the toluene-exposed group compared to the noise-exposed group in Period 2. Furthermore, PLT in the co-exposed group significantly increased compared to the toluene-exposed group in Period 2 (Figure 4(d)).

According to different interactions among multiple stressors (Piggott et al., 2015), this study presents the synergistic and antagonistic effects caused by noise and toluene exposure on the measured hematological parameters (The types of interactions are provided in Tables 1 –4).

Table 1.

Parameter estimates of the generalized estimating equation (GEE) analysis in Period 2 (immediately after exposure).

Parameter	Group 1	Group 2	Group 3	Interaction type
RBC (10⁶/uL)	−0.335^a ± 0.157	−0.502^a ± 0.157	−1.012^b ± 0.168	−Synergism
MCV (fL)	−0.800 ± 0.493	−3.067^b ± 0.720	−3.117^b ± 0.473	−Antagonism
MPV (fL)	−0.133 ± 0.410	−0.283 ± 0.439	−0.283 ± 0.220	−Antagonism
MCH (pg)	0.007 ± 0.007	0.001 ± 0.004	0.011 ± 0.007	+Synergism
HGB (g/dL)	−0.383 ± 0.357	−0.833 ± 0.367	−1.650^a ± 0.481	−Synergism
HCT (%)	−3.250^a ± 0.943	−4.683^a ± 1.346	−8.267^b ± 1.177	−Synergism
PCT (%)	0.122^a ± 0.042	−0.077 ± 0.047	0.086 ± 0.068	−Antagonism
PLT (10³/uL)	179.000 ± 95.573	−86.667 ± 73.599	156.333 ± 90.162	−Antagonism
PDW (log)	−0.006 ± 0.006	0.001 ± 0.007	−0.004 ± 0.004	−Antagonism
RDW-CV (%)	3.767^a ± 1.096	1.333 ± 0.823	2.117 ± 1.258	+Antagonism
RDW-SD (fL)	10.667^b ± 2.890	3.300 ± 2.854	5.517 ± 3.322	+Antagonism
WBC (10³/uL)	−0.250 ± 0.970	−0.078 ± 1.310	1.903 ± 0.978	+Synergism
Lymphocyte (10³/uL)	−0.768 ± 0.915	−1.333^a ± 0.588	0.020 ± 0.898	+Synergism
Neutrophil (10³/uL)	0.977 ± 0.792	1.127 ± 0.964	1.725^a ± 0.721	+Antagonism
Monocyte (10³/uL)	0.103 ± 0.283	0.179 ± 0.355	−0.149 ± 0.328	−Synergism
Eosinophil (10³/uL)	−0.028 ± 0.163	−0.148 ± 0.196	0.115 ± 0.187	+Synergism
Basophile (10³/uL)	0.037 ± 0.044	0.102^b ± 0.026	7.880E-17 ± 0.009	+Antagonism

Group 1: exposure to 100 ± 5 decibel (dB) noise (n = 6).

Group 2: exposure to 1000 ± 50 part per million (ppm) toluene (n = 6).

Group 3: co-exposure to 100 ± 5 dB noise and 1000 ± 50 ppm toluene (n = 6).

^aSignificant changes (p-value <0.05).

^bSignificant changes (p-value <0.001).

Values for these parameters are the β value ± SE (standard error).

Exposure routes: inhalation exposure to toluene and hearing exposure to noise.

RBC: Red blood cells; MCV: Mean corpuscular volume; MPV: Mean platelet volume; MCH: Logarithm of the mean corpuscular hemoglobin; HGB: Hemoglobin; HCT: Hematocrit; PCT: Procalcitonin; PLT: Platelet; PDW (log): Logarithm of platelet distribution width; RDW-CV: Coefficient of variation of red blood cell distribution width; RDW-SD: Standard deviation of red blood cell distribution width; WBC: White blood cells; Monocyte: Logarithm of monocyte; Eosinophil: Logarithm of eosinophil; uL: microliter; fL: femtoliters; pg: picograms; g/dL: grams per deciliter.

Table 2.

Parameter estimates of the generalized estimating equation (GEE) analysis in Period 3 (the 3^rd day).

Parameter	Group 1	Group 2	Group 3	Interaction type
RBC (10⁶/uL)	−0.618^a ± 0.228	−0.857^b ± 109	−1.255^b ± 0.164	−Synergism
MCV (fL)	−0.667 ± 0.729	−1.783 ± 1.098	−1.533^a ± 0.708	−Antagonism
MPV (fL)	−0.667 ± 0.408	−0.300 ± 0.509	−0.617^a ± 0.214	−Antagonism
MCH (pg)	0.014^b ± 0.004	0.015 ± 0.008	0.019^b ± 0.005	+Antagonism
HGB (g/dL)	−0.950 ± 0.422	−1.133^b ± 0.185	−1.933^b ± 0.376	−Antagonism
HCT (%)	−4.933^b ± 1.204	−6.017^b ± 0.940	−8.867^b ± 1.314	−Antagonism
PCT (%)	0.065 ± 0.063	−0.023 ± 0.103	0.019 ± 0.065	+Antagonism
PLT (10³/uL)	159.000 ± 120.009	1.833 ± 148.314	72.167 ± 83.795	+Antagonism
PDW (log)	−0.014 ± 0.007	−0.002 ± 0.008	−0.009 ± 0.004	−Antagonism
RDW-CV (%)	2.567^a ± 0.950	1.200 ± 0.775	3.017 ± 1.819	+Antagonism
RDW-SD (fL)	6.267^a ± 2.698	2.417 ± 2.720	7.683 ± 4.842	+Antagonism
WBC (10³/uL)	−1.202^a ± 0.550	−2.122 ± 1.490	−2.263^a ± 0.999	−Antagonism
Lymphocyte (10³/uL)	−0.717 ± 0.900	−0.832 ± 1.092	−0.723 ± 0.509	−Antagonism
Neutrophil (10³/uL)	−0.853 ± 0.563	−1.190^a ± 0.583	−1.827^a ± 0.871	−Antagonism
Monocyte (10³/uL)	0.388 ± 0.337	−0.455^b ± 0.082	−0.308 ± 0.354	+Antagonism
Eosinophil (10³/uL)	−0.177 ± 0.104	−0.035 ± 0.210	−0.152 ± 0.208	−Antagonism
Basophile (10³/uL)	−0.063 ± 0.047	0.088^b ± 0.021	0.107^b ± 0.021	+Synergism

Group 1: exposure to 100 ± 5 decibel (dB) noise (n = 6).

Group 2: exposure to 1000 ± 50 part per million (ppm) toluene (n = 6).

Group 3: co-exposure to 100 ± 5 dB noise and 1000 ± 50 ppm toluene (n = 6).

^aSignificant changes (p-value <0.05).

^bSignificant changes (p-value <0.001).

Values for these parameters are the β value ± SE (standard error).

Exposure routes: inhalation exposure to toluene and hearing exposure to noise.

Table 3.

Parameter estimates of the generalized estimating equation (GEE) analysis in Period 4 (the 7^th day).

Parameter	Group 1	Group 2	Group 3	Interaction type
RBC (10⁶/uL)	−0.803^a ± 0.367	−1.140^b ± 0.143	−1.157^b ± 0.173	−Antagonism
MCV (fL)	−0.433 ± 0.973	−0.783 ± 1.226	0.083 ± 0.744	+Synergism
MPV (fL)	−0.417 ± 0.446	0.533 ± 0.517	−0.200 ± 0.181	+Antagonism
MCH (pg)	−0.016^a ± 0.006	−0.039 ± 0.044	0.018^a ± 0.005	+Synergism
HGB (g/dL)	−1.967^a ± 0.690	−2.717^a ± 1.093	−1.767^b ± 0.359	−Antagonism
HCT (%)	−5.417^a ± 2.260	−7.350^b ± 1.085	−7.350^b ± 1.249	−Antagonism
PCT (%)	−0.011 ± 0.045	0.002 ± 0.043	−0.033 ± 0.030	−Synergism
PLT (10³/uL)	25.000 ± 89.608	−8.833 ± 77.830	−38.500 ± 36.555	−Synergism
PDW (log)	−0.012 ± 0.008	0.004 ± 0.010	−0.004 ± 0.005	−Antagonism
RDW-CV (%)	3.750^b ± 0.839	3.983^a ± 1.160	3.917^a ± 1.781	+Antagonism
RDW-SD (fL)	9.683^b ± 2.193	10.167^a ± 3.878	9.917^a ± 4.774	+Antagonism
WBC (10³/uL)	−2.548 ± 1.298	−2.572 ± 1.359	−3.530^a ± 1.537	−Antagonism
Lymphocyte (10³/uL)	−1.610 ± 0.971	−2.243^a ± 0.933	−2.680^a ± 0.963	−Antagonism
Neutrophil (10³/uL)	1.073 ± 1.145	−0.328 ± 0.754	−1.047 ± 0.732	−Synergism
Monocyte (10³/uL)	0.155 ± 0.141	0.039 ± 0.296	0.123 ± 0.281	+Antagonism
Eosinophil (10³/uL)	−0.164 ± 0.094	−0.086 ± 0.139	−0.226 ± 0.154	−Antagonism
Basophile (10³/uL)	0.058 ± 0.030	−0.003 ± 0.026	0.017 ± 0.044	+Antagonism

Group 1: exposure to 100 ± 5 decibel (dB) noise (n = 6).

Group 2: exposure to 1000 ± 50 part per million (ppm) toluene (n = 6).

Group 3: co-exposure to 100 ± 5 dB noise and 1000 ± 50 ppm toluene (n = 6).

^aSignificant changes (p-value <0.05).

^bSignificant changes (p-value <0.001).

Values for these parameters are the β value ± SE (standard error).

Exposure routes: inhalation exposure to toluene and hearing exposure to noise.

Table 4.

Parameter estimates of the generalized estimating equation (GEE) analysis in Period 5 (the 14^th day).

Parameter	Group 1	Group 2	Group 3	Interaction type
RBC (10⁶/uL)	−0.097 ± 0.376	−0.840^b ± 0.270	−0.903^b ± 0.191	−Antagonism
MCV (fL)	−0.300 ± 1.176	−2.517 ± 1.818	1.417 ± 0.742	+Synergism
MPV (fL)	0.083 ± 0.418	0.483 ± 0.407	0.150 ± 0.243	+Antagonism
MCH (pg)	−0.020 ± 0.011	−0.003 ± 0.017	0.012^a ± 0.003	+Synergism
HGB (g/dL)	−0.367 ± 0.687	−1.467 ± 0.751	−1.450^b ± 0.359	−Antagonism
HCT (%)	−1.167 ± 2.037	−6.167^a ± 2.197	−5.000^b ± 1.241	−Antagonism
PCT (%)	−0.031 ± 0.032	0.059 ± 0.068	0.041 ± 0.055	−Antagonism
PLT (10³/uL)	45.333 ± 62.807	68.000 ± 95.741	49.000 ± 76.618	−Antagonism
PDW (log)	−0.008 ± 0.006	−0.001 ± 0.006	0.001 ± 0.005	+Synergism
RDW-CV (%)	5.133^b ± 0.944	5.183^b ± 1.439	4.733^b ± 1.318	+Antagonism
RDW-SD (fL)	14.150^b ± 2.428	12.817^b ± 2.810	12.933^b ± 3.713	+Antagonism
WBC (10³/uL)	−1.627^a ± 0.778	−1.213 ± 1.296	−1.297 ± 1.086	−Antagonism
Lymphocyte (10³/uL)	−0.460 ± 0.493	−0.513 ± 0.736	−0.793 ± 0.448	−Antagonism
Neutrophil (10³/uL)	−0.440 ± 0.370	−0.723 ± 0.623	−0.600 ± 0.740	−Antagonism
Monocyte (10³/uL)	−0.106 ± 0.188	0.422 ± 0.249	−0.04 ± 0.457	+Antagonism
Eosinophil (10³/uL)	−0.033 ± 0.142	0.054 ± 0.320	−0.042 ± 0.174	−Synergism
Basophile (10³/uL)	−0.050 ± 0.057	−0.033 ± 0.044	−0.018 ± 0.043	−Antagonism

Group 1: exposure to 100 ± 5 decibel (dB) noise (n = 6).

Group 2: exposure to 1000 ± 50 part per million (ppm) toluene (n = 6).

Group 3: co-exposure to 100 ± 5 dB noise and 1000 ± 50 ppm toluene (n = 6).

^aSignificant changes (p-value <0.05).

^bSignificant changes (p-value <0.001).

Values for these parameters are the β value ± SE (standard error).

Exposure routes: inhalation exposure to toluene and hearing exposure to noise.

Discussion

The present study demonstrated the hematological changes induced by simultaneous and non-simultaneous exposure to noise and toluene. The identification of health effects induced by simultaneous exposure to different physical and chemical pollutants is critical to improving the prevention and diagnosis of various diseases (Rider et al., 2018); therefore, the results of this study can be greatly useful for that purpose. As observed already, the hematologic effects caused by simultaneous exposure to noise and toluene had not been thoroughly studied before, which highlights the importance of this study.

Reduction of RBC by noise exposure in rabbits, as also found in this study, had already been reported in rats and other subjects (Zymantiene et al., 2017; Axelsson and Dengerink, 1987). This study concluded that toluene exposure induced a decrease in RBC, which had already been reported in humans in petrochemical industries (Neghab et al., 2014; Kamal and Malik, 2012). Moreover, co-exposure to noise and toluene showed a decrease in RBC.

This study and previous studies (Zymantiene et al., 2017) showed that exposure to noise caused a decrease in WBC levels. Results of this study indicated that simultaneous exposure to noise and toluene decreased lymphocyte and eosinophil levels and increased basophiles at different post-exposure times. Moreover, exposure to noise and toluene induced a decrease in MCV and PDW levels on different days after the end of the exposure. The differences between the results reported in this study and the findings reported by Mohammadi et al. (2016) can be explained in part by differences in experimental designs and sample types. For example, Mohammadi et al. (2016) excluded workers with underlying diseases, such as cardiovascular and blood disorders, and those who used certain medications. The results of previous studies showed that exposure to construction noise for 1 year decreased HGB levels (Zymantiene et al., 2017). It was also reported that exposure to some organic solvents, such as toluene, decreased HCT and increased HGB levels in petrochemical industries and auto-repair workers (Neghab et al., 2014).

The current study demonstrated that non-simultaneous exposure to noise and toluene induced a decrease in the MCH level, but simultaneous exposure to noise and toluene exhibited an increase in the MCH level. Mohammadi et al. (2016) reported that exposure to industrial noise induced a decrease in MCHC levels in insulator manufacturing workers (Mohammadi et al., 2016). Moreover, increased MCHC was already described by exposure to some organic chemical pollutants such as toluene in petrochemical industries (Neghab et al., 2014).

According to previous studies, stresses such as noise and chemical substances such as toluene can cause aplastic anemia, which is a life-threatening bone marrow disorder and one the most severe causes of blood disorders (Young et al., 2010; Lisiewicz, 1993). The adverse cytogenetic effects of toluene and the induction of micronuclei in bone marrow polychromatic erythrocytes investigated in previous studies indicated the adverse effects of toluene on different blood parameters. It has also been reported that there is a direct correlation between RDW indices and different stressors, which confirms the results of this study (Ahmadi et al., 2019; Neumann et al., 2000).

The results of this study indicated that exposure to noise increased PCT and PLT levels. Qureshi et al. (2002) reported that stress such as noise can decrease PLT levels (Qureshi et al., 2002). The contradiction between some results of this research and previous studies pertaining to MCV, PCT, and PLT levels could be due to the intervention of other chemical and physical agents in industries and factories; therefore, it is essential to conduct further studies on the hemotoxic effects of exposure to toluene or controlled noise to delineate more specific alterations.

The elevation of ROS levels by exposure to noise (Ohlemiller et al., 1999; Nicotera et al., 1999) and toluene (Revilla et al., 2007; Mattia et al., 1991) has already been reported. ROS can enhance macrophages, WBCs, and immunoglobulin, and induce inflammatory reactions by generating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that stimulates the production of inflammatory cytokines (Gloire et al., 2006). Previous studies have reported that exposure to toluene (Tellez-Martinez et al., 2019) and psychological stressors (Curtin et al., 2009) separately can enhance inflammatory cytokines. Therefore, the immune system inhibits the permanent generation of inflammatory cytokines early during the exposure by secreting specific leukocytes that can express several anti-inflammatory cytokines such as IL-10 and inhibits the generation of NF-κB to stabilize inflammatory responses (Bogdan et al., 1991; Ding et al., 1993; Kasten et al., 2010; Said et al., 2010). As observed by a study on the immune changes caused by exposure to noise at 90 dB, acute exposure to noise increased immune responses, even though chronic exposure to noise suppressed cellular and humoral immune functions (Zheng and Ariizumi, 2007). As such, it was expected that WBC should increase until the final days of exposure due to exposure to noise and to toluene. Moreover, WBC would increase from the early days of the exposure until the last days of exposure by simultaneous exposure to noise and toluene. On the other hand, inflammatory cytokines have direct interactions with the PCT level (Reinhart et al., 2000; Whang et al., 2000).

Previous studies revealed that stress increases oxidative stress, and noise and toluene are physical and chemical stressors (Zheng and Ariizumi, 2007; Mattia et al., 1993a, 1993b; Rapido, 2017). Oxidative stress can induce hemolysis in RBCs (Perrone et al., 2012), which can decrease HGB, MCH (Waggiallah and Alzohairy, 2011), and RBC levels (Rapido, 2017). Moreover, physical stressors such as noise (Miura et al., 2002) and chemical stressors such as toluene (Lopreato et al., 2003; Rea et al., 1984) raise serotonin levels; consequently, increasing serotonin levels can elevate the PLT level (Lopez-Vilchez et al., 2009).

According to different interactions among multiple stressors (Piggott et al., 2015), this study provides synergistic and antagonistic effects caused by noise and toluene exposure on the hematological parameters measured during different days after the end of exposure (Tables 1 –4).

Limitations

Limited exposure duration and using the same noise and toluene specifications were some limitations of this study. Epidemiological studies on people co-exposed to noise and toluene in various worker populations should be conducted to improve these novel results. Therefore, caution should be exercised when applying the statistical analysis of the reported data.

Conclusion

This study demonstrated that simultaneous or non-simultaneous exposure to noise and toluene can alter different hematological parameters in New Zealand white rabbits. For some parameters, noise and toluene induced synergistic effects, whereas for other parameters, they induced antagonistic effects. Antagonistic effects might present the homeostasis mechanism in the body. It means that the defense mechanism in the body tries to cease serious reactions after exposure to noise and toluene. Although the results demonstrated both synergistic and antagonistic effects after exposure to noise and toluene, we cannot conclude that noise or toluene definitely diminish each other’s toxicity.

These results can enhance our knowledge of environmental and occupational toxicology in terms of the combined effects of harmful stressors. However, further studies need to prove the definite hematotoxicity induced by simultaneous exposure to noise and toluene.

Footnotes

Acknowledgements

We appreciate Tabriz University of Medical Sciences.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

The study was certified by the Ethics Committee of Tabriz University of Medical Sciences (research ethics certification code: IR.TBZMED.REC.1396.953).

ORCID iDs

Amirreza Abouee-Mehrizi

Yahya Rasoulzadeh

Mehran Mesgari-Abbasi

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