In order to evaluate the health effects of long-term occupational BTX co-exposure, we established BEPWC cohort. We observed a significant decrease in most hematologic parameters after four-year follow-up. We found a positive association of benzene with the decline in monocyte counts and a negative association of toluene with the decline in MCHC. The associations of BTX components with the decline in hematologic parameters were mostly significantly stronger in subjects with higher baseline hematologic parameters, males, drinkers, and overweighted subjects. gWQS regression models found that BTX had combined effects on the decline in monocyte counts, RBC counts, and hemoglobin concentrations. These findings provided further evidence of complex hematological effects of long-term occupational exposure to BTX components. Finally, we estimated the BMD and BMDL for each BTX component to proposed some REL based on their hematological effects.
The most commonly-used exposure assessment methods in occupational population are external environment monitoring and internal exposure biomarker detection. However, BTX components and their metabolites in the biological fluids of exposed subjects have relatively short half-lives (Carrieri et al. 2006), which makes them less suitable for assessing long-term exposure levels. Although personal sampling is a more precise exposure assessment method, it is relatively difficult to be long-termly adopted in a cohort study with a relatively large sample size. Furthermore, Lee et al. (2006) have reported that there was no difference in BTX levels between personal and area sampling methods. With these theoretical and practical considerations, we chose area sampling method to measure long-term TWA concentrations for BTX components in workplaces of BEPWC participants. We found that the annual average TWA concentrations of all BTX components were relatively stable. Thus, we calculated the individual BTX CED levels based on long-term average TWA concentrations and work years. We also performed a pilot substudy and observed a significant correlation between the CED indices (represented by benzene CED levels) and commonly-used biomarkers (represented by urinary SPMA) in BEPWC cohort.
The present study found that most hematologic parameters were significantly decreased after four-year follow-up. The plausible mechanisms might be the cumulative exposure to hazard factors with hematotoxicity and the aging-related functional attrition of hematopoietic stem cells (HSCs). Petrochemical industry is a major source of multiple hazardous and toxic air pollution. Mounting evidence have linked increased risk of hematological malignancies, especially leukemia, with petrochemical industrial activities and residential petrochemical exposure (Jephcote et al. 2020; Lin et al. 2020;). Some cross-sectional studies have also explored the influences of petrochemical industry on early hematological effects, mainly reflected by the discordant differences of hematologic parameters in exposed individuals when compared to controls (Chen et al. 2019; Tsai et al. 2004). Khuder et al. (1999) conducted a small cohort study in 105 workers from a petroleum company, and observed all complete blood counts, except for WBC counts, were significantly decreased during the follow-up period. Our larger cohort study also observed some reduction in hematologic parameters, further highlighting the importance of monitoring hematologic parameters in health surveillance and protection for petrochemical workers. Furthermore, the chronological and biological age increased during the follow-up period. Hematopoiesis declines during aging, which emerges as the integrative consequence of multiple types of damage, such as decrease in cell cycle activity of HSCs, accumulation of DNA damages, telomere shortening, and increased levels of inflammatory signaling (Bousounis et al. 2021; López-Otín et al. 2013), The deregulation of HSC homeostasis may result in adverse hematological effects, such as an increased incidence of amenia and hematological malignancies (Bousounis et al. 2021; López-Otín et al. 2013). Our findings might provide further epidemiologic evidence of aging-related decline in hematopoiesis, manifested as decreased hematologic parameters. Intriguingly, we observed significant sex differences in the magnitude of declines in hematologic parameters. It is widely known that important biological and behavioral differences are existed between females and male, which affect the incidence, prognosis, and mortality of many widespread diseases, including hematological malignancies (Ben-Batalla et al. 2019).
Considering that BTX components are important toxic substances in the releases from petrochemical industry, we preliminarily explored their associations with the decline in hematologic parameters. We observed a positive association of benzene with the decline in monocyte counts and a negative association of toluene with the decline in MCHC. Benzene is the most hazardous and striking BTX compound which has been widely-recognized to induce hematotoxicity in humans for over 100 years (McHale et al. 2012). One representative manifestation of benzene-induced hematotoxicity is the reduction in all types of leukocytes (including monocytes), even in a low dose (<1ppm) (Lan et al. 2004; McHale et al. 2012). The present study had also found a positive association of long-term occupational benzene exposure with the decline in monocyte counts. However, the widely-reported relations of benzene with WBC counts were not found in this longitudinal analysis. This may be due to the relatively short follow-up period during which the effects of benzene might not yet be present. In additions to benzene, the influences of toluene on hematologic parameters have also drawn more and more attentions in recent years (Cakmak et al. 2020; Chen et al. 2019; Doherty et al. 2017). The cross-sectional studies conducted by Cakmak et al. (2020), Chen et al. (2019), and Doherty et al. (2017) have observed some significant but inconsistent associations of toluene with several hematologic parameters, such as hemoglobin concentrations and the counts of WBC, lymphocytes, RBC, and platelets. In the present study, we also found a negative association of benzene with the decline in MCHC. The findings from the previous studies and our research collectively suggested that toluene may also have the potential to induce hematotoxicity. However, the existing evidences regarding its hematological effects are relatively limited and inconsistent (ATSDR 2017), and further investigations are warranted to look at the hematological effects of toluene in more depth and verify our findings.
Effect modification analysis showed that the associations of BTX exposure with the decline in hematologic parameters were mostly stronger in subjects with higher baseline parameters. Two meta-analyses of randomized clinical trials have also observed some similar findings (Brunström and Carlberg 2018; Navarese et al. 2018). Brunström and Carlberg (2018) found that treatment to lower blood pressure was associated with a reduced risk for death and cardiovascular disease when baseline systolic blood pressure ≥140 mm Hg, while the treatment effect was limited when baseline systolic blood pressure < 140 mm Hg. Similarly, Navarese et al. (2018) also observed that LDL-C lowering therapy was associated with a reduced total mortality with higher baseline LDL-C levels, while this relationship was not present with lower baseline LDL-C. These findings from the previous studies and our study suggested that subjects with higher baseline levels might be more sensitive to the effects of external factors (including toxic substances and drugs), while those with lower baseline values might be less likely to experience any further decreases, probably due to homeostasis mechanisms. We also found that the association of BTX exposure with the decline in hematologic parameters were mostly stronger in males. Worldwide data have shown that compared with females, males are both at increased risk and have a poorer prognosis for leukemia (Ben-Batalla et al. 2019). Our findings further proved that males might be more predisposed to BTX-associated hematotoxicity. The possible causes for these phenomena include differences in other environmental exposures, lifestyle, endogenous hormones, sex chromosomes, epigenetics, and the complex multidirectional interactions between these factors (Ben-Batalla et al. 2019). Besides, Wang et al. (2021) found that under the same environmental exposure, the benzene metabolites in females' urine were higher than males, which may be due to the higher biotransformation ability and faster detoxification of benzene in females than males. Furthermore, the present study also observed stronger associations of BTX exposure with the decline in hematologic parameters in overweighted subjects with BMI ≥24 kg/m2. Due to their lipophilicity, BTX components mainly accumulate in the fat-enriched tissues. Therefore, fat content in the body might affect the distribution, metabolism, and eventually the toxicity of BTX components (Zhang et al. 2020). Taken together, these results of effect modification analysis provided some clues that can be used for more detailed risk assessment. However, further researches are needed to verify our findings of effect modification and determine the underlying mechanisms.
In addition, we used gWQS regression to model the associations of a mix of three BTX components with the decline in hematologic parameters. We revealed that benzene, toluene, and xylene had combined effects on the decline in monocyte counts, RBC counts, and hemoglobin concentrations, further providing some new epidemiological evidence of the existence of complex joint toxic actions among BTX components in producing hematotoxicity. The joint toxic actions among BTX components are of great concern for a long time (ATSDR 2004). For instance, high amounts of toluene have been shown to inhibit benzene metabolism and subsequent myelotoxicity (Andrews et al. 1977), while lower concentrations of toluene have been suggested to enhance benzene genotoxicity and confounded its effects on many metabolic enzymes and blood cell populations (Bird et al. 2010; Robert Schnatter et al. 2010). Competitive metabolic interactions are believed to be the most plausible mechanism of BTX combined effects, as they are all known substrates for CYP2E1 (ATSDR 2004). The effect of CYP2E1 induction of one BTX component might be masked by other competing substrate(s) at higher co-exposures, but at lower co-exposures the induction provides for more BTX components to be metabolized to toxic metabolites (Bird et al. 2010). Complex combined effects among BTX components on the decline in hematologic parameters were also observed in the BEPWC participants who had been co-exposed to lower concentrations of BTX components. Although the underlying mechanisms are still not quite clear, our study further pointed to the need to scrutinize levels of all BTX compounds in the occupational environment and consider their co-exposure effects in the risk assessment.
Exposure standards are developed to protect the public and the employee from toxic health effects of harmful substances and activities. One of the most crucial processes in exposure standard setting is dose-response relationship assessment which provides the technical information on which to base the setting of standards for “safe” exposure. Recently, BMD estimation method is widely accepted by regulatory agencies and scientific community to determine the “point of departure” (POD) for a specific dose-response relationship and derive human health guidance values (Qing et al. 2021). We also used BMD method in the present study to propose some REL for BTX components based on their hematological effects. Determination of endpoints is the first step for BMD calculations. However, there are currently no unified widely-acknowledged definitions for hematological damage. Many studies, including the present study, have evaluated the associations of BTX components and hematologic parameters, but they have reported many inconsistent results, even for benzene (ATSDR 2007a, b, 2017). Given the complex and inconsistent findings for the hematological effects of BTX components, it might be very hard for us to decide which hematologic parameter should be selected to define hematological damage. Furthermore, using only one parameter (such as WBC counts) to define hematological damage might exclude the endpoints with the potential of having the most sensitive effect for risk assessment applications from the BMD analysis (EPA 2012). Thus, it might be more appropriate to define hematological damage based on a serial of the hematologic parameters, not only one parameter. In the present study, we selected night hematologic parameters which have clinical relevance to chronic exposure to toxic chemicals and bone marrow problems as the parameters of primary interest. In order to increase the specificity and reduce the false positive rate, only the subjects with at least two “abnormal” parameters were classified as the cases of hematological damage. Furthermore, in order to increase the sensitivity, we defined hematological damage not only according to the hematologic parameter levels detected in follow-up stage, but also based on their four-year decline. We considered it as “abnormal” if the levels of at least two selected parameters (except for MPV which has no related national standards) in follow-up stage were below the lower limits (not both lower and upper limits) of normal range in Chinese adults. We also considered it as “abnormal” if the magnitude of the decline in at least two selected parameters higher than the upper 5% percentile in population of the same sex (not in the total population). The main reasons were as follows. Firstly, it is commonly believed that BTX components, especially benzene, are associated with decreased numbers of blood cell types (ATSDR 2007a, b, 2017). Secondly, the results in the present study, although many of which were insignificant after FDR adjustment, also suggested that BTX components might be positively associated with the four-year decline in most hematologic parameters. Furthermore, the magnitude of decline in parameters and their associations with BTX components showed significant sex differences in the present study.
Based on the calculated BMDL and up to 40 years of working duration, we proposed REL-TWA for benzene, toluene, and xylene as 0.0390 mg/m3 (about 0.0125 ppm), 0.0331 mg/m3 (about 0.0088 ppm), and 0.0578 mg/m3 (about 0.0122 ppm), respectively. These REL-TWA proposed in the present study were much lower the current regulatory or advisory exposure limits, such as Permissible Exposure Limits-TWA (PEL-TWA) established by U.S. Occupational Safety and Health Administration (OSHA), Permissible Concentrations-TWA (PC-TWA) in China, Threshold Limit Values-TWA (TLV-TWA) recommended by American Conference of Governmental and Industrial Hygienists (ACGIH), and RfC established by EPA. Zhang et al. (2016) also estimated BMD based on only WBC counts in benzene-exposed workers of shoe factories. With BMR levels at 0.1 in the risk of reduced WBC counts, the BMDL for benzene CED levels was calculated as 1.37 ppm×year (about 4.37 mg/m3×year) (Zhang et al. 2016), which was a little higher than our estimated BMDL (1.559 mg/m3×year). These differences might be due to the different definition of hematological damages. However, as the BMD models couldn’t include covariates, the results of BMD estimation would be vulnerable to confounding bias which may limit the modeling of human data to some extent (EPA 2012). Furthermore, considering that the exposure standards are mostly determined by a trade-off or balancing of the assessed risks and costs of reducing exposure levels, further researches are needed to verify our findings and assess the availability of these REL-TWA of BTX components estimated in the present cohort study.
The present study had several major strengths. First, our participants were petrochemical workers who had been regularly exposed to BTX-rich emissions for more than one year, with their occupational BTX source and levels showing little fluctuation. Thus, we calculated BTX CED levels to evaluate their long-term exposure doses, rather than the internal exposure biomarkers which may reflect recent exposure. Secondly, we used BMD estimation method to propose REL based on some reasonable and comprehensive criteria of hematological damage. However, some limitations in the present study should also be noted. Firstly, in order to eliminate the influences of baseline health status on our results, we intentionally selected the BEPWC participants from workers without any serious diseases or any obviously abnormities in baseline physical examination. Furthermore, these participants had been exposed to BTX components for, on average, 18.75 years prior to the baseline stage. Thus, it was likely to result in healthy worker selection and survivor effects that might bias our results. Secondly, the present study was conducted in an occupational population, and it is uncertain whether our findings could be extrapolated to the general population. Moreover, although our BEPWC participants were intentionally selected from the workplaces where BTX compounds were always the primary occupational hazards, they may be also co-exposed to other hazards in their working and living environments which may also cause hematotoxicity. Despite we had adjusted factory location and main lifestyle factors (such as smoking and drinking status) in the statistical analysis to control their confounding effects as a whole, further studies are still warranted to evaluate their influences on our results.