Introduction

BTX are common pollutants in occupational environments and originate from sources such as vehicle exhaust, cigarette smoke, petrochemicals, pharmaceutical products, and various solvents used in industries such as paint manufacturing, rubber products, and adhesive production1. At ambient temperature, BTX is a volatile liquid with a characteristic aromatic odour that disperses easily into the air, where it can enter the body through the skin or respiratory tract, leading to significant health impacts. Urinary S-PMA, HA, and MHA are commonly used as biomarkers for the biological monitoring of benzene, toluene, and xylene exposure2. Biomonitoring of BTX can provide additional information to air monitoring, reflecting both the pulmonary and the dermal uptake of benzene. Benzene is rapidly metabolized and cleared from the blood and is often monitored in the urine as unmetabolized benzene or its metabolites3. In regard to inhaled toluene, 15–20% is excreted through exhalation, with the remaining 80% converted to HA and eliminated via the urine after 7 hours4. Xylene is absorbed and oxidized to methylbenzoic acids, conjugated with glycine to form MHA, and then eliminated through the urine5. Additionally, creatinine is a byproduct of muscle metabolism and is mainly excreted through the kidneys. Urinary solute concentrations are easily affected by water reabsorption in the kidney; therefore, the creatinine concentration in urine is usually used to normalize the urinary concentration of metabolites6. The production and use of benzene in China are increasing gradually. In 2026, the petroleum benzene capacity is expected to reach 25.875 million t/a7.

BTX exposure is a growing public health concern. Benzene is haematotoxic and carcinogenic and has been classified as a human carcinogen by the International Agency for Research on Cancer (IARC) since 1982, whereas toluene and xylene were listed as Group III carcinogens by the IARC in 2017. Long-term high-concentration benzene exposure increases the risk of leukaemia, aplastic anaemia, and other diseases8,9. With strengthened management in China, most workplace BTX concentrations are below occupational exposure limits10, but low-concentration exposure still poses risks of neurological, haematologic, immunologic, and cardiovascular damage11. Although the harmful effects of benzene have been studied more frequently, exposure to this single substance is uncommon in the modern industrial world; toluene and xylene are regular companions, and their additive effects have not yet been adequately assessed12.

Several studies have noted that low-concentration BTX exposure can increase blood pressure in workers13,14, suggesting that low-concentration BTX exposure may be a risk factor for increased blood pressure. In 1969, researchers discovered superoxide dismutase (SOD), which can scavenge reactive oxygen species (ROS)15, and subsequently, the free radical theory centred on oxidative stress was a popular topic of research in the biomedical community. BTX exposure increases ROS production in the body, leading to oxidative stress, and current epidemiological studies have revealed that oxidative stress is associated with various pathological processes in cardiovascular disease16,17,18,19. SOD and MDA are the most commonly used biomarkers to evaluate oxidative stress20. SOD is a key antioxidant enzyme that maintains the oxidative‒antioxidative balance, whereas MDA is a final product of lipid peroxidation, serving as a marker of oxidative damage, with levels positively correlated with the extent of damage. Previous population studies have shown that changes in SOD and MDA levels are associated with an increased incidence of chronic diseases21,22. Furthermore, studies have confirmed that elevated blood pressure is linked to an imbalance between oxidative and antioxidant capacity23,24,25. However, whether oxidative stress plays a potential role in the elevation of blood pressure due to BTX exposure is unknown.

In this study, a cross-sectional study was conducted to investigate whether oxidative stress plays a mediating role in the relationship between exposure to BTX and blood pressure among workers of a petroleum refining plant in Hainan Province. Physical examinations, questionnaires, and laboratory tests, as well as monitoring the concentration of BTX in the workplace, were conducted to provide a basis for further revealing the mechanism of elevated blood pressure induced by exposure to BTX.

Methods

Statement: The experiments in this study were conducted at the laboratory of Hainan Medical University, with all experiments approved by the respective laboratories. All the experiments were performed in accordance with the relevant guidelines and regulations.

Study design and participants

In June 2022, we adopted a cross-sectional study and conducted a questionnaire survey to investigate workers from a petroleum refinery in Hainan Province, China. The questionnaire, designed by the research team, covered sociodemographic information, lifestyle behaviours, and occupational information. Additionally, professional physical examination institutions conducted occupational health examinations for employees, primarily focusing on blood pressure, body mass index (BMI), and other relevant tests. Blood pressure measurement: After the workers had rested quietly for 10 min, blood pressure was measured three times via an electronic sphygmomanometer, with a 30-second interval between each measurement. The average of the SBP and DBP readings was taken as the final result. Those exposed to BTX were categorized into the exposed group, which included individuals engaged in benzene operations, oil storage and transportation, and chemical analysis. The exposure group was defined on the basis of the findings from the field investigation. Conversely, workers in administrative, management, and logistical positions were selected as the control group. Finally, 615 workers exposed to BTX were selected as the exposed group, whereas 226 workers from the same enterprise were chosen as the control group. The inclusion criteria were age ≥ 18 years and continuous working time (work tenure) ≥ 1 year in the current workplace. The exclusion criteria were as follows: less than 1 year of work tenure, those who refused to participate in the survey, and a missing questionnaire information rate exceeding 20%. Written informed consent was obtained from the participants, and our study was approved by the Ethics Committee of Hainan Medical University (No: HYLL-2022-247).

Laboratory testing

BTX detection: According to the Specifications of Air Sampling for Hazardous Substances Monitoring in the Workplace GBZ 159–201426 and Determination of Aromatic Hydrocarbons in the Air of Workplace GBZ/T 160.42–200727, the concentration of BTX in operating positions during the normal production of the enterprise was sampled and detected on site. Main monitoring points: Benzene operations: toluene distillation, adsorption separation, bottom of the isomerization adsorption tower, third-floor platform of the circulating benzene tank, inspection position of the circulating benzene heater blower, external control room of the styrene unit, and other locations. The oil storage and transportation facilities included the central control room of the aromatic product tank area, tank area, berth valve area of the port operations centre, external control room of the port operations centre berth, and other locations. The chemical analysis facilities included the oil analysis room of the quality control centre, the chromatography analysis room of the quality control centre, and other locations. Urine sample collection and detection of BTX metabolites: Midstream urine samples (10 mL) were collected from participants and promptly refrigerated before transportation to the laboratory. High-performance liquid chromatography‒tandem triple quadrupole mass spectrometry (HPLC‒MS/MS) was used to detect signature metabolites of BTX in urine, including S-PMA for benzene, HA for toluene, and MHA for xylene metabolism. MHA comprises three isomeric forms: 2-MHA, 3-MHA, and 4-MHA. A random 10% sample of the population underwent urine analysis for BTX metabolites, including 62 individuals from the exposure group and 23 individuals from the control group. Moreover, creatinine correction was used to adjust for BTX metabolites in urine. Peripheral blood serum collection and oxidative stress indicator testing: Blood was collected via an EDTA-K2 anticoagulation tube to obtain fasting peripheral blood samples (3 mL/person). The blood was centrifuged to separate the serum, which was stored at -80 °C until oxidative stress analyses were performed. For the determination of the serum SOD and MDA levels, a superoxide dismutase assay kit (water-soluble tetrazolium salt-1 (WST-1) method) and a malondialdehyde assay kit (thiobarbituric acid (TBA) method) (Nanjing Jiancheng Bioengineering Institute, Article No. A001-3-2, A003-1-2) were used, and the measurements were performed according to the instruction manuals of the kits.

Definition of indicators

The BMI was calculated via the following formula for Chinese residents: BMI = weight (kg)/Height2 (m2). The classifications included underweight, BMI < 18.5 kg/m2; normal, 18.5 kg/m2 ≤ BMI < 24.0 kg/m2; overweight, 24.0 kg/m2 ≤ BMI < 28.0 kg/m2; and obese, BMI ≥ 28.0 kg/m228. Smoking: Smoking was defined as having 6 months or more of continuous or cumulative smoking with at least 1 cigarette per day. Alcohol use: those who consumed alcohol at least once a week in the past year were defined as alcohol use. The working model: regular day shift refers to workers working in different shifts for 24 h without stopping; shift work refers to workers working on a fixed day shift29. Occupational stress: The Chinese version of the effort–reward imbalance (ERI) scale was used to assess the level of occupational stress30. An ERI > 1 indicates occupational stress, and an ERI ≤ 1 indicates no occupational stress. Low-concentration BTX: the BTX exposure concentrations of subjects in the exposure group met the values stipulated in China’s national occupational health criteria: Occupational exposure limits for hazardous agents in the workplace—Part 1: Chemical hazardous agents GBZ 2.1–2019 Revision No. 131 and Occupational exposure limits for hazardous agents in the workplace—Part 1: Chemical hazardous agents GBZ 2.1–201932. The permissible concentration‒time weighted average (PC-TWA) values of benzene, toluene, and xylene in the working environment are 3 mg/m3, 50 mg/m3, and 50 mg/m3, respectively, and the permissible concentration‒short-term exposure limits (PC-STELs) are 6 mg/m3, 100 mg/m3, and 100 mg/m3, respectively.

Statistical analysis

The questionnaire results and physical examination data were double-entered via EpiData 3.0 software. Nonnormally distributed data are presented as medians (interquartile ranges), whereas normally distributed data are expressed as the means ± standard deviations. The distributions of the oxidative stress markers SOD and MDA were log10-transformed. Multiple linear regression was employed to analyse the relationships between low-concentration BTX exposure and SOD, MDA, SBP, and DBP. The correlation between blood pressure levels and oxidative stress levels was analysed via partial correlation tests. Bootstrap mediation analysis was performed via the SPSS PROCESS V3.5 macro developed by HAYES33, with mediation Model 4 (simple mediation model). R language 4.3 and SPSS 22 were utilized for the aforementioned analyses. A P value of less than 0.05 was considered statistically significant.

Results

The concentrations of benzene, toluene, and xylene (BTX) in the occupational environment and the levels of BTX metabolites in the urine of workers

The results of the occupational environmental detection indicated that PC-TWA concentrations for benzene were < 0.6 mg/m3, those for toluene were < 1.2 mg/m3, and those for xylene were < 3.3 mg/m3. The PC-STEL concentrations for benzene were < 0.6 mg/m3, those for toluene were < 1.2 mg/m3, and those for xylene were < 3.3 mg/m3. None of these concentrations exceed the national occupational exposure limit. The levels of BTX metabolites in the urine of the two groups of workers are shown in Table 1. The concentration of the benzene metabolite S-PMA in the urinary samples in the low-concentration BTX-exposed group was significantly greater than that in the control group (1.77 (1.11 ~ 2.39) vs. 1.10 (0.98 ~ 2.13) mg/g Cr, respectively, P < 0.05). The concentration of the toluene metabolite HA in the urine samples in the low-concentration BTX-exposed group was significantly greater than that in the control group (131.90 (57.32 ~ 249.22) vs. 54.66 (34.05 ~ 107.43) mg/g Cr, respectively, P < 0.05). The concentrations of the xylene metabolites 3-MHA and 4-MHA in the urine samples from the low-concentration BTX-exposed group were significantly greater than those in the control group (0.05 (0.03 ~ 0.10) vs. 0.03 (0.02 ~ 0.06) mg/g Cr and 0.04 (0.02 ~ 0.10) vs. 0.02 (0.01 ~ 0.06) mg/g Cr, respectively, P < 0.01 for both). The urinary concentrations of the xylene metabolites 2-MHA, 3-MHA and 4-MHA were significantly greater in the smoker group than in the nonsmoker group (0.07 (0.04 ~ 0.15) vs. 0.03 (0.02 ~ 0.07) mg/g Cr, 0.09 (0.04 ~ 0.17) vs. 0.03 (0.02 ~ 0.06) mg/g Cr and 0.08 (0.03 ~ 0.17) vs. 0.03 (0.02 ~ 0.06) mg/g Cr, respectively, P < 0.01 for both). A correlation analysis was conducted between urinary concentrations of BTX metabolites and blood pressure levels. The results revealed a positive correlation between the concentration of the benzene metabolite S-PMA in workers’ urine and diastolic blood pressure levels (r = 0.265, P < 0.05), as shown in Table 2.

Table 1 Concentrations of BTX metabolites in the urine of the study participants [M(P25 ~ P75)]
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Table 2 Correlation analysis between the concentrations of BTX metabolites in the urine and blood pressure levels.
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Demographic, occupational, and lifestyle characteristics of the participants

The demographic, occupational and lifestyle characteristics of the participants are shown in Table 3. A total of 841 participants were enrolled. Most participants were 35 years old or younger and composed 70.3% of the sample. Those with more than 5 years of work tenure constituted 55.1% of the sample. A total of 87.2% of the participants were men. Additionally, 27.7% of the participants reported being smokers, whereas 49.0% were alcohol users. Notably, low-concentration BTX exposure accounted for more than two-thirds (73.1%) of the sample.

Blood pressure levels of participants with different characteristics

The blood pressure levels of the study participants across different demographic groups are presented in Table 3. The levels of SBP and DBP for all participants were 117.12 ± 11.76 mmHg and 77.34 ± 8.73 mmHg, respectively. Workers were stratified on the basis of various characteristics, and blood pressure levels were compared among these groups. Gender disparities were evident in both SBP and DBP levels (P < 0.05 for both), with men exhibiting higher SBP and DBP values than women. Significant differences in SBP and DBP levels were observed across different age brackets (P < 0.05 for both), with elevated levels noted in the > 35 years age group. Stratification by work tenure revealed statistically significant differences in both SBP and DBP levels (P < 0.05 for both), with higher levels observed among those with > 5 years of work tenure. Distinct BMI categories also exhibited significant disparities in SBP and DBP levels (P < 0.05 for both), with the obese subgroup having the highest values. Compared with nonsmokers, smokers presented significantly greater SBP levels (P < 0.05), whereas no significant difference in DBP levels was detected between smokers and nonsmokers (P > 0.05). Both SBP and DBP levels were significantly greater among alcohol users than among nonusers (P < 0.05 for both). The DBP level was 77.77 ± 8.57 mmHg in the low-concentration BTX-exposed group and 76.17 ± 9.07 mmHg in the control group, with a statistical significance (P < 0.05). However, the SBP level was 117.29 ± 11.95 mmHg in the low-concentration BTX-exposed group and 116.64 ± 11.24 mmHg in the control group, but the difference was not statistically significant (P > 0.05).

Table 3 Blood pressure levels in different population groups (‾x ± s).
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Comparison of oxidative stress markers between the exposed group and control group

The levels of oxidative stress markers in both the exposed and control groups are depicted in Fig. 1. Statistical analysis revealed significant differences in the levels of SOD and MDA in the serum between the exposed and control groups (P < 0.05 for both). Specifically, the exposed group presented lower serum SOD activity [8.02 (6.69, 10.07) U/mL] than did the control group [8.57 (7.32, 10.74) U/mL], whereas the serum MDA concentration in the exposed group [4.57 (3.17, 7.27) nmol/mL] was greater than that in the control group [4.20 (2.92, 5.92) nmol/mL].

Fig. 1
figure 1

Violin diagrams of oxidative stress markers in the exposed group and control group. **P < 0.01, SOD superoxide dismutase, MDA malondialdehyde. In the violin plot, the first dashed line is the third quartile, the second dashed line is the median, and the third dashed line is the first quartile.

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Correlations between low-concentration benzene, toluene, and xylene (BTX) exposure and blood pressure and oxidative stress markers

After collinearity diagnostics were conducted for the independent variables intended for inclusion in the multiple linear regression analysis, the tolerance values for all the independent variables were greater than 0.1, and the variance inflation factors were all less than 10, indicating the absence of multicollinearity among the independent variables. Following adjustment for potential confounding factors in the multiple linear regression model, the analysis of the associations between low-concentration BTX exposure and blood pressure, as well as oxidative stress levels, is presented in Table 4. The analysis revealed that the DBP levels in the low-concentration BTX exposure group were significantly greater than those in the control group (β = 1.363, 95% CI 0.088 ~ 2.639) (P < 0.05). However, no significant difference in SBP levels was detected between the low-concentration BTX exposure group and control group (P > 0.05). Furthermore, the serum SOD activity was significantly lower in the low-concentration BTX exposure group than in the control group (β = − 0.037, 95% CI − 0.060 to − 0.013) (P < 0.05), whereas the serum MDA concentration was significantly greater in the low-concentration BTX exposure group than in the control group (β = 0.066, 95% CI 0.022 ~ 0.110) (P < 0.05).

Table 4 Multifactor analysis of the effects of low-concentration BTX exposure on blood pressure and oxidative stress biomarkers among occupational workers [β (95% CI)]
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Correlation analysis between oxidative stress levels and blood pressure levels

Partial correlation analysis was performed while controlling for the variables of sex, age, work tenure, BMI, smoking, and alcohol use among workers. The results of the analysis are presented in Table 5 and indicate a positive correlation between MDA and DBP (rs = 0.115, P < 0.05).

Table 5 Correlation analysis between oxidative stress levels and blood pressure levels.
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Mediation model

Mediation analysis revealed that BTX exposure had a significant indirect effect on DBP through the mediating variable MDA. The 95% CI for the indirect effect was 0.0561 ~ 0.4250, indicating a mediating effect. Conversely, the 95% CI for the direct effect was − 0.1284 ~ 2.4220, suggesting that the direct effect was not statistically significant. These findings suggest that MDA fully mediated the relationship between BTX exposure and DBP (see Table 6).

Table 6 Mediating effects of MDA on the BTX-induced increase in DBP.
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Discussion

Our findings suggest that occupational exposure to low concentrations of BTX may lead to increased DBP levels, reduced SOD activity, and elevated MDA content among workers. Additionally, our analysis revealed a positive correlation between MDA levels and DBP. Notably, the results of the mediation analysis indicate that MDA serves as a complete mediator of the increase in DBP among workers exposed to low concentrations of BTX.

Industries associated with oil and gas extraction are significant contributors to hydrocarbon emissions, including volatile organic compounds (VOCs), aldehydes, olefins, and phenols. Consequently, workers within these sectors face escalated health hazards34,35, with prolonged occupational exposure to such compounds linked to a diverse spectrum of detrimental health outcomes. This burden adds substantially to the already formidable global disease burden36. The pollution of the environment by BTX aromatics and its impact on human health has become a public safety and health issue that urgently needs to be addressed. In China, the petroleum processing industry presents greater occupational health risks associated with benzene exposure than do sectors such as chemical product manufacturing, specialized equipment manufacturing, wood processing and products, and pharmaceutical manufacturing37. Currently, most occupational workers are conscious of personal protection, but exposure to low concentrations of BTX is often unavoidable. The results of this study revealed that the concentrations of S-PMA, HA, 3-MHA, and 4-MHA in the urine of workers in the low-concentration BTX exposure group were significantly greater than those in the control group, with statistically significant differences. Although the concentration of 2-MHA was greater in the exposure group than in the control group, the difference was not statistically significant, which may be attributed to the following reasons. First, the sample size was insufficient. The limited sample size may have reduced the statistical power, making it difficult to detect actual differences. Second, differences in metabolic pathways exist: Xylene metabolites are not limited to 2-MHA; other metabolic pathways, such as those producing 3-MHA and 4-MHA, may play a more prominent role in different populations. Given that cigarettes contain BTX compounds, this study compared the metabolites of BTX between smokers and nonsmokers. The results revealed that the levels of xylene metabolites were greater in smokers than in nonsmokers. In subsequent research, smoking was included as a control variable in the statistical analysis. Research indicates that even exposure to benzene levels below the occupational exposure limit can adversely affect organismic health, possibly due to synergistic interactions among benzene compounds in mixed exposures38. However, this study revealed that the DBP level of workers increased with increasing concentrations of the benzene metabolite S-PMA in the urine under exposure to low concentrations of BTX. Studies have shown that long-term exposure to low-concentration BTX can lead to an increase in the incidence of hypertension and an abnormal detection rate of systolic blood pressure in workers14. Furthermore, other studies have reported elevated DBP levels in workers exposed to low concentrations of BTX13. The findings of our study underscore a significant association between occupational exposure to low concentrations of BTX and heightened DBP levels among workers. Prior investigations have suggested that this correlation may arise from disturbances in nitric oxide biosynthesis, thereby exacerbating blood pressure39.

Long-term exposure to low concentrations of BTX can induce an imbalance in oxidative antioxidants within the body. Under normal physiological conditions, endogenous oxidants and antioxidants maintain equilibrium, yet excessive oxidant production disrupts this balance, leading to oxidative stress. This stress generates an abundance of reactive oxygen species, causing damage to proteins, lipids, and nucleic acids and potentially resulting in DNA oxidation and modification40. SOD and MDA are commonly utilized as biomarkers to evaluate oxidative stress status20. Research has demonstrated that exposure to low concentrations of BTX significantly increases MDA levels in workers while concurrently altering SOD activity41. Consistent with prior findings, our study revealed a similar pattern: compared with the control group, the group exposed to low concentrations of BTX presented diminished serum SOD activity and elevated MDA concentrations. These observations suggest that exposure to low concentrations of BTX may disrupt the balance of oxidative antioxidants within the body.

Hypertensive patients exhibit a disruption in the oxidative antioxidant equilibrium within their bodies. In 1990, researchers reported elevated levels of cellular H2O2, O2, and lipid peroxidation products in patients with essential hypertension compared with the general population, as well as in normotensive individuals with a familial predisposition to hypertension42,43. Growing evidence underscores an imbalance between oxidative and antioxidant capacities in hypertensive patients, with oxidative stress intricately linked to hypertension onset and progression. ROS generated by vascular endothelial and smooth muscle cells can induce sustained blood pressure elevation and vascular damage44,45. The vascular phenotype of hypertension is closely intertwined with oxidative stress, and the levels of its biomarkers influence the extent of end-organ damage46. Additionally, oxidative stress can induce hypertension by fostering renal vasoconstriction and perturbing sodium homeostasis47. Clinical investigations have revealed positive correlations between SBP and diastolic blood pressure DBP levels with oxidative markers and inverse correlations with antioxidant levels in hypertensive patients48. Several studies have highlighted a significant increase in serum MDA levels with increasing blood pressure and a concomitant decrease in serum SOD activity49,50. In our study, DBP levels increased with increasing MDA concentration, suggesting a potential association between elevated DBP and oxidative stress. Mechanistically, this could be attributed to long-term hypertension-induced vascular endothelial damage, which promotes inflammatory cell adhesion, activation, and phagocytosis, thereby generating excess oxygen radicals51. Furthermore, our findings suggest that MDA serves as a complete mediator in the relationship between exposure to low concentrations of BTX and DBP. These results imply that low concentrations of BTX may impact DBP levels indirectly through MDA. Consequently, there appears to be a potential aetiologic association between exposure to low concentrations of BTX and MDA and DBP.

This research enhances the existing body of occupational epidemiological literature regarding the associations between exposure to BTX and blood pressure and oxidative stress. Furthermore, these findings elucidate the mediating role of MDA in the increase in blood pressure induced by BTX exposure. These results have significant implications for hypertension prevention in this population, including suggestions for dietary modifications and the incorporation of antioxidant supplements. Consistent with the principles of the Mediterranean diet, this nutritional approach offers antioxidants that may effectively mitigate the effects of BTX exposure52. Notably, this study represents an inaugural investigation into MDA as a mediating factor in the increase in DBP attributed to low-concentration BTX exposure in an occupationally exposed population.

Our study has the following limitations. First, workplace BTX exposure was monitored via area sampling rather than more precise personal sampling, potentially resulting in a less accurate assessment of participant exposure levels. Second, given the presence of various other toxic and hazardous substances in petroleum refining workplaces, this study did not account for the potential health effects resulting from combined exposure to multiple environmental pollutants. Third, blood pressure levels are influenced by numerous factors, including the living environment and personal habits, which were not fully accounted for in this study because of limitations in survey data. Finally, because of the poor power of causal arguments in cross-sectional studies, while our findings contribute to elucidating the relationships among low-concentration BTX exposure, oxidative stress markers, and blood pressure, the establishment of a causal relationship remains incomplete. However, we provide ideas for follow-up studies through cross-sectional studies. Future long-term longitudinal studies are warranted to explore this relationship further; therefore, further cohort studies are essential to elucidate the causal relationships involved.