Association of IL-8 and CXCR2 with AST/ALT Ratio in Liver Abnormalities Screening during Oxidative Stress Injury Caused by VCM

Liver impairment caused by VCM has been linked to irreversible damage such as fibrosis, necrosis, hepatocellular carcinoma, and liver angiosarcoma. However, the ability to detect abnormalities during initial phase have not been achieved so far. Thus, this study aimed to investigate the effect of interleukin 8 (IL-8) and C-X-C chemokines 2 (CXCR2) on screening for a VCM-exposed group (n = 227) from a PVC manufacturing factory compared to a control group (n = 110) in Tianjin City in 2020 with influence factors evaluation. Ambient concentrations of VCM and health archives from 2012 to 2018 were collected for establishing the dose-effect trend. A cross-sectional survey in 2020 was performed to measure TDGA, IL-8, CXCR2, 8-OHdG, SOD, GPX, CAT, MDA, and ROS levels. Results indicated a continuous increased incidence on liver abnormalities despite a fluctuated downward trend in cumulative time-weighted average (CTWA) VCM concentrations over the years. ALT, AST, and AST/ALT ratio all contributed to liver abnormalities that contained fatty liver, liver calcification, and liver cysts, IL-8 and CXCR2 correlated with each other strongly and showed significant associations with oxidative stress markers, even AST/ALT ratio. IL-8 (>1547 µg/m3) or CXCR2 (<139 µg/m3) influenced the AST/ALT ratio through reciprocal interactions under oxidative stress injury, CXCR2 (>222 µg/m3), working years of 21 to 30 (a) and 11 to 20 (a), TDGA (>1.52 mg/L), alcohol consumption, smoking habit, and a less sleeping duration of <4 h per day would also be potential factors affecting the AST/ALT ratio. In conclusion (1) even with decreased VCM concentrations in PVC manufacturing factories liver abnormalities that contained fatty liver, liver calcification, and liver cysts could still occur due to oxidative stress injury with involvement of IL-8 and CXCR2. The status of protective measure and appropriate mask types also play a role; (2) the AST/ALT ratio could be a specific indicator for detecting abnormalities when combined with liver B ultrasonography results before impairment altered from bad to worse; and (3) factors such as definite medication history, fully broken protective facilities, alcohol consumption, less sleeping duration, inappropriate mask types, and longer working years could also influence AST/ALT ratio alterations through complex interactions.


Introduction
Vinyl chloride monomer (VCM) is a volatile organochlorine liquid that typically exists in a gaseous form at room temperature for high volatility.VCM is used primarily for the production of polymer polyvinyl chloride (PVC), with specialties such as water-proofng, anticorrosion, insulation, and plasticity for producing pipes, coating of wire, building materials, and so on [1].
Occupationally, individuals are primarily exposed to ambient VCM through respiratory inhalation.In the liver, VCM is metabolized via the oxidation of CYP2E1, resulting in the formation of a highly reactive intermediate called 2chloroethylene oxide.Tis intermediate spontaneously rearranges into 2-chloroacetaldehyde and is further converted to 2-chloroethanol.Finally, the detoxifcation process involves the action of glutathione and aldehyde dehydrogenase, which convert it into thiodiglycolic acid (TDGA) and N-acetyl-2-(2-hydroxyethyl) cysteine for convenient excretion [2].It has been proven that high levels of VCM can induce severe health damage.Tis damage is characterized by acroosteolysis, respiratory irritation on the alveolar epithelium, ataxia, dizziness, and liver impairment.Tese health efects often involve toxicant-associated steatohepatitis (TASH), peripheral fbrosis, necrosis, hepatocellular carcinoma (HCC), and liver angiosarcoma (ASL) [3].
Technically, acetylene hydrochlorination and ethylene oxychlorination are the two dominant techniques for PVC production.Among them, acetylene hydrochlorination still holds a majority share in the domestic market due to abundant raw materials (calcium carbide ore), low investment requirements, strong technological improvement capability, and independent intellectual property rights that align with the development needs of the coal-electricitychemical industry.
However, concerns have been raised about the potential health risks associated with VCM exposure, particularly regarding the development of liver abnormalities.Despite eforts to minimize VCM-related health damage, research on the adverse efects of VCM exposure has been ongoing.For example, a case study conducted in 1974 [4] revealed a prevalence of ASL among workers engaged in manual cleaning of PVC polymerization reactors at a plant in Louisville, Kentucky, USA.Te study found that ASL occurred after a long latency period (median 36 years) and was predominantly observed in workers with high cumulative VCM exposures (>1000 parts per million (PPM)-years), especially those exposed to ≥10,551 ppm-years who had a hazard ratio of 73. 6.
In another study conducted in Porto Marghera, Italy [5], among 1658 workers in a VCM/PVC plant, baggers exposed to VCM were found to have a standardized mortality ratio (SMR) of 1.73 for lung cancer (90% confdence interval 0.93∼3.21), and the SMR ratio between baggers and control posts was 2.31 (90% CI: 1.15∼4.61).Tese fndings highlight the need to investigate whether liver abnormalities can still develop even when current exposure concentrations are below the hygiene limits, as well as the identifcation of typical imaging results that can facilitate the prompt screening and monitoring of liver abnormalities.
Given that VCM was ranked fourth on the Centers for Disease Control and Prevention's Agency for Toxic Substances and Disease Registry Substance Priority List in 2006 [6], indicating its potential hazards.It has also been classifed as a Group I carcinogen by the International Agency for Research on Cancer (IARC) [7,8], based on animal and occupational epidemiological studies conducted in 2007 and 2012, respectively, which link VCM exposure to HCC and ASL with tangible evidence.
Considering that China's VCM production capacity reached 23.53 million tons in 2018 and is expected to reach 25.93 million tons by 2023 [9], it becomes crucial to prioritize the initial screening for abnormal symptoms or specifc indicators, as the health status of workers can often be neglected in high-content and demand environments.One cost-efective method widely used in clinical practice for screening liver abnormalities is liver B ultrasonography.Tis method allows for the identifcation of conditions such as fatty liver, alcoholic cirrhosis, steatohepatitis, periportal fbrosis with cysts, and even HCC by assessing liver edge bluntness, liver parenchyma coarseness, liver surface nodularity, inferior vena cava irregularity and narrowness, echogenicity, distribution of calcifcation, and hepatic cysts [10,11].
Interleukin 8 (IL-8) is a peptide consisting of 72 amino acids that can be secreted by fbroblasts, endothelial cells, epithelial cells, monocytes, macrophages, and cancer cells.It typically signals through two G-protein-coupled receptors, CXCR1 and CXCR2, which belong to the C-X-C chemokine receptor group.Numerous studies have suggested that IL-8 and CXCR1/2 signaling play a role in recruiting neutrophil granulocytes to sites of infammation; additionally, they contribute to tumor growth by promoting angiogenesis and immune resistance [12].
Moreover, it has been confrmed that the IL-8-CXCR1 signaling pathway primarily promotes cancer cell proliferation in breast cancer, ovarian cancer, and pancreatic cancer [13].On the other hand, the IL-8-CXCR2 coalition is known to be involved in angiogenesis and the recruitment of neutrophils to the tumor microenvironment [14].For example, recent studies have shown that the IL-8-CXCR2 axis plays a signifcant role in the progression of various liver diseases such as alcohol or nonalcohol-related liver disease, hepatitis (HBV/HCV), fbrosis, and cirrhosis [15].Additionally, liver hepatocytes and resident immune cells can sense changes in the environment and signals in the bloodstream [16].Once activated by antigens or external insults, CXCR2 and IL-8 are released from the liver to recruit neutrophils, leading to neutrophil-derived oxidative bursts and release of cytotoxic granules, ultimately causing hepatotoxicity and hepatocellular death [17,18].In this regard, it is speculated that alterations in the IL-8-CXCR2 interaction may contribute to liver impairment through involvement in oxidative stress injury.Terefore, it is of interest to investigate whether IL-8 and CXCR2 play potential roles or regulate specifc indicators during the initial stage of liver impairment under exposure to VCM.

Journal of Toxicology
In order to investigate this hypothesis, the objective of this study was to examine the impact of IL-8 and CXCR2 on liver abnormalities and identify potential correlations with health indicators during the initial screening for liver impairments.Furthermore, this study will also analyze other factors that may be crucial in identifying key risk factors in workplace environments.Te ultimate goal is to provide efective intervention measures and establish regulated health surveillance for individuals exposed to VCM.

Study Design and Population.
A cross-sectional survey that focused on the VCM exposed population was conducted in 2020 in a large-scale PVC manufacturing factory (referred to as G) located in Bin Hai New Area, Tianjin City, China.During the initial stage, a total of 227 workers from 8 major positions in the VCM manufacturing facilities were identifed as the exposed group.In comparison, 110 operators from a local canned food processing plant (referred to as H) with no history of chemical hazard exposure were selected as the control group based on specifc inclusion and exclusion criteria.
All subjects from either group G or group H had to meet the following requirements: (1) age between 20 and 60 years with no gender limitations; (2) a minimum length of employment in the current factory of at least 1 year and a minimum current working duration of 3 months; (3) involvement in tasks including but not limited to patrol or feld operation.Conversely, workers were excluded if they met any of the following criteria: (1) a history of hepatitis virus B or C infection; (2) clinical diagnosis of alcoholic fatty liver, alcoholic liver cirrhosis, or other liver impairments caused from alcohol abuse, trauma, or complicating diseases; (3) a past or present medical history of chronic renal or liver disease, cardiovascular and cerebrovascular diseases such as hypertension, type 2 diabetes, heart disease, and hepatobiliary calculus; (4) incomplete information or lack of health data; and (5) refusal to provide biological specimens or sign informed consent.
Te ethical approval for this study was obtained from the Medical Ethics Committee of the National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention in Beijing, China (NIOHP202119).Participants who expressed their willingness to take part in the study were informed about the purpose and detailed implementation steps and were required to sign a written informed consent.Furthermore, all methods conducted in this study were in accordance with the relevant guidelines and regulations of the Helsinki Declaration and were approved by the academic board of the National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention.

Manufacturing Process Investigation in G and H.
Factory G is primarily involved in the production of VCM, with an annual production capacity of 800,000 tons, which is further processed into PVC at a rate of 1,000,000 tons per year by using the acetylene hydrochlorination technique.Te overall process can be described in the following steps: (1) acetylene is generated through a chemical reaction between water and broken calcium carbide; (2) VCM is synthesized by reacting acetylene with hydrogen chloride under the catalytic infuence of mercuric chloride (HgCl 2 ); (3) fnally, PVC is produced through the VCM polymerization carried out under high temperature and pressure.In contrast, Factory H specializes in the production of canned food, including yellow peaches, pineapples and other fruits.Te processes involved in the production can be described as follows: (4) raw materials such as fruits and vegetables are stripped, cleaned and cut; (5) Te cans or jars are flled using automatic machines and any gases are exhausted; (6) the cans are sealed with iron caps and subjected to sterilization; and (7) packaging and quality examination are conducted before the products are shipped.Figures 1(a) and 1(b) display the process fow diagrams for Factories G and H, respectively.

Questionnaire Survey.
During the physical examination process in 2020, a questionnaire interview was conducted in a face-to-face manner.All participants who voluntarily agreed to take part in the study were provided with information about the purpose and gave their consent.Te interviewers were trained in advance to ensure objective inquiry and minimize subjective bias.Te questionnaire covered various aspects, including: gender (male/female); age (∼30/∼45/∼60a); working years (∼10/∼20/∼30a); medication history (yes/no)]; smoking habits (yes/no); alcohol consumption (yes/no); sleeping duration (∼4/∼6/∼8 h); weekly exposure time (∼20/∼40 h); mask types [antipoison/ antiparticulate/disposable ones; frequency of wearing [always/certain times (especially on feeding raw materials, cleaning up working environment or sampling)]; mask replacement frequency [once per day/infrequently (switching frequency that lower than 3 times per week)]; and protective facility status (normally operating, temporary suspending, and fully broken).Tese details were collected to ensure a thorough understanding of the participants' backgrounds and work environments.

Health Data Collection.
A comprehensive collection of health data and medical records was carried out, encompassing various parameters.Tese included white blood cell counts (WBC) ranging from 4.0 to 10.0 × 10 9 /L, red blood cell counts (RBC) ranging from 4.5 to 5.5 × 10 12 /L for males and 3.5 to 5.0 × 10 12 /L for females, hemoglobin levels (HGB) ranging from 120 to 160 g/L for males and 110 to 150 g/L for females, blood platelet counts (PLT) ranging from 100 to 300 × 10 9 /L.Furthermore, hepatic function indexes were measured, including alanine aminotransferase (ALT) levels ranging from 5 to 40 U/L, glutamyl transpeptidase (c-GGT) levels ranging from 0 to 50 U/L, aspartic transaminase (AST) levels ranging from 8 to 40 U/L, and AST/ALT ratio should be less than 1.15.Hemobilirubin (TBIL) levels ranged from 3.4 to 17.1 μmol/L, total protein (TP) levels ranged from 60 to 80 g/L, globulin (Glb) levels ranged from 20 to 30 g/L, and Journal of Toxicology albumin (Alb) levels ranged from 35 to 50 g/L.Additionally, blood biochemistry assessments measured total cholesterol (TC) levels, which should be less than 5.2 mmol/L, and triglyceride (TG) levels, which should be less than 1.7 mmol/ L. Tese measurements were conducted biennially from 2012 to 2018.In 2020, other indices such as alkaline phosphatase (ALP) levels ranging from 4 to 150 U/L, absolute neutrophil count (ANC) ranging from 2.0 to 4.0 × 10 9 /L and fasting blood glucose (FBG) ranging from 3.9 to 6.
In the context of this study, the notation used for various parameters is as follows: C represents the ambient concentration of VCM in milligrams per cubic meter (mg/m 3 ), C 0 represents the concentration of VCM within the measured samples in micrograms per milliliter (μg/mL), V 0 represents the standardized sampling volume in liters (L), and D represents the desorption efciency in percentage (%).Te entire set of operating steps followed the guidelines outlined in GBZ/T 300.78-2017 (determination of toxic substances in workplace air-Part 78: vinyl chloride, dichloroethene, trichloroethene, and tetrachloroethene) [19].Following the completion of the secondary incubation, the reagent was discarded and the washing steps were repeated fve times.Chromogenic reagent A and B were added sequentially to each well, and the plate was kept at 37 °C away from light for 10 minutes to allow the chromogenic reaction to occur.Once color development was observed, the reaction was terminated by adding 50 μL of stop bufer to each well.Te absorbance of the wells was measured at a wavelength of 450 nm using a microplate reader (Multiskan ™ FC, Termal, USA).Te content of each index was then calculated using a linear regression equation derived from the relevant standard series.
2.8.Urinal TDGA Detection.TDGA content was determined using solid phase extraction ion chromatography, following the specifed procedures [20].Te operational steps were as follows: Sequentially, 2 mL of chromatographically pure methanol (Fisher Scientifc, USA) and 2 mL of deionized water (>1822 MΩ•cm) were added to C18 solid phase extraction columns (200 g and 3 mL per column, Agilent, USA).Te columns were then further activated through natural leaching with cleaned residential liquid.Urine specimens (15 mL volume per column) were centrifuged at 3000 r/min for 10 minutes.From the resulting supernatant, 2.5 mL was extracted and titrated into a 15 mL centrifuge tube (CORNING, USA) with deionized water to scale.After shaking and mixing for 10 minutes, 2.0 mL of the diluted urine was passed through activated C18 columns.Te fltering liquid was fully collected and passed through a needletype fltering head (pore size 0.22 μm, Zenten, Germany) once more.Te collected fltering liquid was used for loading.Detailed steps for chromatographic column analysis (LonPac ASl9, 250 mm × 4 mm) were followed, the mobile phase for column protection to IonPae AGl9 was KOH liquid, which was created by an in-line generator with leaching liquid.Te gradient leaching pattern (with a fow rate of 1.0 mL/min) consisted of 20 mmol/L for 0-10 minutes, 20-30 mmol/L for 10-20 minutes, 30-60 mmol/L for 20-30 minutes, and 60 mmol/L for 30-36 minutes.Te current for the suppressor was set at 150 mA and the automatic injection quantity was limited to 25 μL.Standard series were prepared using the following steps: Precisely weighing 50 mg of TDGA standard material (Dr.Ehrenstorfer GmbH, Germany) and making it into a standardized stock solution of 1000 μg/mL.Diluting the stock solution further to prepare a standard series of 100 μg/ mL (with concentrations of 0, 0.05, 0.10, 0.50, 2.50, 10.00, and 50.00 μg/mL).Te injection quantity was limited to 25 μL, and results were determined by retention time (21.13 min) and quantifed based on peak area.Equation ( 2) is represented as follows: where C represents the mass concentration (μg/mL) of TDGA in urine.C 0 represents the mass concentration (μg/ mL) of TDGA detected through solid phase extraction ion chromatography.Te coefcient of 6 represents the dilution factor of 6 times.1(a), 1(b), 1(c), and 1(d).

Multiple Logistic Regression of Health Data between
Groups in 2020.Subsequently, a comparison was made between health data of the two groups in 2020 to identify any diferences that could potentially contribute to specifc diagnostic categories.In Figure 6(a), signifcant diferences were observed in WBC, PLT, TP, Glb, Alb, TG, TC, ALT, c-GGT, and AST/ALT ratios between the groups.Specifcally, in the exposed group, ALT, AST, and AST/ALT ratio played noteworthy roles in "liver calcifcation" and "liver cysts," while ALT specifcally contributed to "fatty liver" ( * * P < 0.05).Furthermore, TG, TC, ANC, TP, Glb, and Alb were found to play roles either in "fatty liver" or in "liver calcifcation" ( * * P < 0.05), as shown in Figures 6(b) and 6(c).On the other hand, in the control group, TG and TC played signifcant roles across all categories, while TP and Glb had minimal contributions to "fatty liver" and "liver calcifcation" ( * * P < 0.05).Additionally, the AST/ALT ratio and TP showed other contributions to "liver cysts" ( * * P < 0.05) instead.Tus, it appeared that ALT, AST, and AST/ALT ratio demonstrated signifcant contributions to abnormality categories under VCM exposure, while TG, TC, ANC, TP,         Figure 5: Curve charts (a-d) presented content diferences toward WBC, RBC, HGB, PLT, ANC, FBG, TG, TC, TP, Glb, Alb, ALT, AST, ALP, c-GGT, and AST/ALT ratio among categories of "fatty liver," "liver calcifcation," and "liver cysts" compared to "normal" in the exposed group at 2020; curve charts (e-h) equally displayed expression levels of indicators in the control group at 2020, as * * * * indicated P < 0.001, * * meant P < 0.05.

Journal of Toxicology
Glb, and Alb may not be specifc indicators.Some studies showed that they could be indicators for nonalcoholic fatty liver disease (NAFLD) caused by a high-fat diet (HFD).For example, Lang et al. [22] study on mice exposed to VCM and supplied with an HFD demonstrated that VCM exposure at levels considered safe (OSHA <1 ppm) could worsen experimental NAFLD and led to liver impairment due to secondary insults, such as decreased mitochondrial function and ER stress.Tese efects can afect mechanisms related to VCM-enhanced HFD-induced liver injury.Te signifcant decrease in mitochondrial activity and subsequent depletion of adenosine triphosphate likely contributed to increased liver injury, elevated production of reactive oxygen species, ultimately led to an increased hepatocyte necrosis.

Odds Ratio of Liver B Ultrasonography Results in 2020.
Te risk of specifc liver abnormalities in diferent exposure groups was assessed by using odds ratios (OR).To calculate the OR, the numbers of workers with liver abnormalities (a) and without liver abnormalities (c) were compared to ones with liver abnormalities (b) and without liver abnormalities (d) in the "normal" group.Te OR values for workers in the low-, medium-, and high-exposure groups were found to be 1.6-fold, 2.4-fold, and 2.6-fold higher for "fatty liver," respectively, 8.2-fold, 12.6-fold, and 14.2-fold higher for "liver calcifcation," and 6.1-fold, 5.9-fold, and 8.6-fold higher for "liver cysts," respectively, as compared to those in the "normal" group.Te most signifcant increase was observed in the incidence of "liver calcifcation," followed by "fatty liver" and "liver cysts" in order.In addition, a signifcant  Journal of Toxicology diference in population size was observed among the different exposure groups by using the chi-square analysis (X 2 � 44.32, P < 0.001), as shown in Figure 8.All these suggested that risks of developing liver calcifcation, liver cysts, and fatty liver in the exposed group were closely associated with VCM exposure levels as the increased concentration of VCM may serve as a contributing factor to the rising incidence of liver abnormalities.Guardiola et al. [23] investigated a combined efect of diet-induced liver injury and VCM exposure in mice.Teir study found that an imbalance in the hepatic oxidative defense system induced by toxicants could lead to hepatic infammation.Liver defense enzymes showed that VCM exposure signifcantly depleted hepatic superoxide dismutase 3 (SOD3) and heme oxygenase 1 (HO1), but had insignifcant efects on SOD1, SOD2, CAT, nuclear factor erythroid-derived 2 (NRF2), and MDA levels.Tese suggested that levels of IL-8 and CXCR2 in the exposed group were elevated in response to increased VCM concentrations, which indicating the extent of oxidative stress injury and changes in antioxidative enzymes.
In the control group, it was noted that MDA (β � −0.463, 95% CI � −7.800∼-1.011)and ROS (β � 0.330, 95% CI � 0.078∼6.301)involved in correlation with 8-OHdG content ( * * P < 0.05), and the contribution made from CXCR2 (β � 0.011, 95% CI � 0.067∼0.438)was negligible, as Table 3 revealed.At this point, it could be inferred that IL-8 and CXCR2 in the exposed group obviously refected the extent of oxidative stress injury and were subsequently related to alterations of AST/ALT ratio in a linear manner under VCM exposure.Tus, the oxidative stress injury might be a key mechanism that intervened in the normal ranges of AST/ALT ratio, where IL-8 and CXCR2 might play potential roles.

Interactive Analysis of Liver B Ultrasonography Results
between Groups in 2020.Based on the information presented in Tables 1 and 2, it appeared that variables such as alcohol consumption, working years, mask replacement frequency, mask types, sleeping duration, gender as well as medication history all contributed to liver abnormalities for varying extents.It was worth to explore whether there were any interactive links among these variables.To facilitate binary category analysis, the liver B ultrasonography results, originally classifed as normal, fatty liver, liver calcifcation, and liver cysts, were regrouped into "normal" and "abnormal" types.In the exposed group, it was found that content of 8-OHdG (<85, 85∼148, >148 µg/m 3 ) interacted with medication history (F � 3.376, P < 0.05) and sleeping duration (F � 5.168, P < 0.05), respectively.Specifcally, for workers with a defnite medication history, content of 8-OHdG exceeding 148 µg/m 3 was 0.97 times higher than that of <85 µg/m 3 (95% CI � 0.196∼1.823,P < 0.05).Furthermore, for 8-OHdG content exceeding 148 µg/m 3 , workers with an average sleeping duration of 4-6 hours had a 0.83fold higher risk as compared to those with a sleeping duration of 6-8 hours (95% CI � 0.252∼1.406,P < 0.05).Content of TDGA (<0.33, 0.33∼1.52,>1.52 mg/L) also interacted with the status of protective facilities (F � 3.035, P < 0.05) and medication history (F � 5.448, P < 0.05).Particularly, for workers who considered protective facilities to be in normal operating status, risks associated with TDGA content of 0.33∼1.52mg/L were 1.3 times higher than those associated with >1.52 mg/L (95% CI � 0.402∼2.199,P < 0.05).When TDGA content exceeded 1.52 mg/L, risks for workers identifying temporary suspension were 1.1 times higher than those considering it as normal operating (95% CI � 0.393∼1.801,P < 0.05).Additionally, for workers with a defnite medication history, risks associated with TDGA content exceeding 1.52 mg/L were 1.7 times higher than those associated with <0.33 mg/L (95% CI � 0.528∼2.917,P < 0.05) as presented in Figures 11(a), 11(b), 11(c), and 11(d).In the control group, the only interactive link observed was between mask type and status of protective facilities (F � 2.645, P < 0.05).For workers who usually wore antiparticulate masks, risks with ones who regarding temporarily suspended facilities were 0.68 times higher than for those confrmed as fully functional (95% CI � 0.241∼1.636,P < 0.05), as Figure 11(e) presented.It could be inferred that high levels of 8-OHdG (>148 µg/m 3 ) interacted with a definite medication history and shorter sleeping duration (4-6 hours) to contribute to liver abnormalities as oxidative stress injury.Furthermore, TDGA content (>1.52 mg/L) also interacted with the status of protective facilities on temporary suspension and a defnite medication history, particularly when VCM concentration reached relatively high levels.Inadequate protective facilities and inappropriate mask types might also increase the risks of liver abnormalities through reciprocal interactions.

Interactive Analysis of the AST/ALT Ratio between
Groups in 2020.Based on fndings presented in Figures 5  and 6, it was evident to note that the AST/ALT ratio played a signifcant role in determining the binary categorization of liver as "normal" or "abnormal" in consecutive years from 2012 to 2018, as well as in specifc abnormality categories such as "normal," "fatty liver," "liver calcifcation," and "liver cysts" in 2020.It was of interest to explore any potential interactions between IL-8, CXCR2, 8-OHdG, TDGA, and variables from questionnaire and how these interactions correlated with the AST/ALT ratio through a general linear model under pairwise comparison analysis.

Discussion
Numerous studies have indicated that high levels of occupational exposure to VCM will lead to liver damage, which is often characterized by hepatocellular injury and functional disturbances.By the time liver abnormalities are detected through imaging or abnormal indicators, it may already be too late for urgent clinical treatment [24].Tis study observed various changes in liver function indicators (ALT, AST, and AST/ALT ratio), results from liver B ultrasonography tests (including fatty liver, liver calcifcation, and liver cysts), detected indices for infammatory factors such as IL-8 and CXCR2, as well as indices concerning oxidative stress injury.Tese fndings suggested that these factors could potentially be utilized for screening liver abnormalities prior to liver impairment.
Te results of the retrospective survey revealed that liver abnormalities caused by VCM exposure were consistently present across diferent job posts.Levels of ALT, AST, AST/ ALT ratio, and c-GGT were higher in workers with "abnormal" symptoms compared to those with "normal" symptoms, even improvement measures on practical reductions in VCM concentration had taken place.Furthermore, the composition ratios (r%) of "abnormal symptoms" indicated a continuous tendency towards liver impairment over years.Tese fndings suggested that the reduction in VCM concentration did not prevent liver abnormalities from progression and development.In fact, improvement measures were already being implemented in VCM manufacturing facilities to reduce VCM concentration from a preliminary survey.Previous results from our study [25] showed a signifcant decrease in abnormal hepatic symptoms in the majority of posts at the PVC factory (Y) that employed the acetylene hydrochlorination technique, as compared to another factory that using the ethylene oxychlorination technique in Tianjin City.Improvements, such as enhanced ventilation and air-tightness, also showed notable efects on health indicators related to fatty liver and other hepatic symptoms in both factories (Y and Z), including ALT, AST, and c-GGT levels.Tese fndings indicated that changes in these indices partially refected the status of liver impairment and the efectiveness of the improvement measures.In particular, the volatile gas released from VCM, especially originated from open observation ports, was controlled through emergency ventilation facilities with increased exhaust air and ventilation air changing rates.Binary logistic regression analysis from 2012 to 2018 and multiple logistic regression analysis in 2020 all demonstrated that indicators of ALT, AST, and AST/ALT ratio could serve as prominent indicators for screening liver abnormalities in the initial phase.Various domestic and international studies have shown that the aminotransferase levels or AST/ALT ratio could be potential biomarkers for liver functional impairment, including the incidence of liver cancer and abnormalities in metabolic functions, such as hyperlipidemia and hyperglycemia [21].In fact, the concept of the aspartate transaminase/alanine transaminase (AST/ ALT) ratio is initially proposed for the study of hepatitis etiology and is commonly used to diferentiate diferent causes of liver disease, such as fatty liver.It is considered an efective biomarker for liver disease screening, especially when the AST/ALT ratio exceeds the limit of 1.15, which is a clinical and preventive standard for cardiovascular diseases, various cancers even type 2 diabetes mellitus [26].A high AST/ALT ratio (>1.15) has also been associated with predicting poor prognosis in nonmetastatic renal cell carcinoma.For instance, a study with 9,946 participants conducted by the BaiYun community Health Service Center in Taizhou, Jiangsu Province, China (2015-2019), revealed signifcant correlations between elevated AST/ALT ratio and increased mortality risks (OR � 1.495, P < 0.05, 95% CI � 1.003-2.228)after adjusting for demographic and behavioral variables [27].Te AST/ALT ratio has also been identifed as an independent risk factor for forecasting population health risks, positive associations have been observed between blood pressure and AST/ALT ratio with diastolic blood pressure.Furthermore, the AST/ALT ratio has been advocated for cancer development.For example, Li et al. found that a higher AST/ALT ratio was an independent predictor of 1-year mortality in polymyositis-/dermatomyositis-associated interstitial lung disease, and it was associated with poor outcomes in renal cell carcinoma, head and neck carcinoma, oral and oropharyngeal carcinoma, and other malignant cancers [28].Based on these fndings, the AST/ALTratio could be considered as an efective biomarker for identifying liver abnormalities caused by VCM during the initial phase, supported by abundant evidence.Furthermore, the 2020 VCM concentration detection revealed a strong correlation between higher concentrations in the exposed group and their relevant TDGA content.Specifcally, posts occupied by aggregated operators, stripping operators, polymerization cleaners, maintenance repairers, and feld samplers had concentrations within the range of 50% to 1 fold OEL or exceeded 1 fold OEL.Tis indicated that workers were potentially at risk of health, and requirements to reduce ambient VCM exposure levels immediately were urgent.During feld investigations, several vulnerabilities in VCM production facilities were identifed, particularly in the maintenance of protective measures, operation of airtight techniques, and implementation of warning notices.Tese three factors were the main contributors to occupational health risks, particularly, it usually involved in insufcient emergency ventilation systems, malfunctioning ventilation equipment, inadequate indoor air distribution, improper setup of exhaust hoods, uncovered observation ports, unsealed valves or cover plates, and absence of warning lines and enclosures.All of these systematically afected the difusion and spatial distribution of VCM concentration, ultimately compromising the health status of workers.Subsequently, odds ratio (OR) values gradually increased when grouping specifc categories related to liver calcifcation, liver cysts, and fatty liver.Tis fnding suggested a hypothesis that the increased VCM concentration among workers may be a primary cause of the rising incidence of liver abnormalities.
Detection on IL-8, CXCR2, and other oxidative stress injury indices revealed that increased exposure to VCM could elevate the levels of IL-8 and CXCR2 and intensify their correlation among workers through oxidative stress injury mechanisms.Studies conducted by Chen [29] and Nguyen NT [30] have shown that IL-8 typically binds tightly to CXCR2 and this IL8-CXCR2 axis plays a pivotal role in liver infammation induction.It recruits neutrophils to infection or injury sites, where they primarily clear pathogens, cellular debris, alarmins, and metabolic waste.However, this process can also induce infammation injury and lead to hepatocyte death.Once neutrophils reach the tissues, CXCR2 activation triggers the release of granule enzymes and reactive oxygen species (ROS), which aid in pathogen elimination and up-regulate IL-8 expression.Te infltration and accumulation of immune cells have been found to correlate signifcantly with chronic infammation in the liver, based on these, IL-8 and its ligand CXCR2 were selected as the breakthrough point.
Additionally, multiple logistic linear analyses suggested that IL-8, CXCR2, and 8-OHdG could infuence the AST/ ALT ratio under VCM exposure.Interaction analysis also indicated that high levels of IL-8 (>1547 µg/m 3 ) or low levels of CXCR2 (<139 µg/m 3 ) were associated with changes in the AST/ALT ratio under oxidative stress injury.Moreover, other interactions were observed between CXCR2 (>222 µg/m 3 ) and longer working years of 21 to 30 (a) or 11 to 20 (a), as well as high TDGA content (>1.52 mg/L) and alcohol consumption.It could be assumed that long-term sleep disorders, irregular insomnia, and factors such as spiritual pressure, heavy workload, disturbed biological clocks due to long working years, and even occupational  psychological stress induced by environmental noise or high temperatures all probably contributed to liver impairment and might exacerbated occupational health risks.Interaction analysis results for liver ultrasonography tests, categorized as abnormal and normal, showed that high levels of 8-OHdG (>148 µg/m 3 ) interacted with a defnite medication history and shorter sleeping duration (4∼6 hours) in contributing to liver abnormalities.Additionally, TDGA content (>1.52 mg/L) was noted to interact with the presence of protective facilities during temporary suspension and a defnite medication history, particularly when VCM concentration reached relatively high levels.Inadequate protective facilities and the use of inappropriate mask types also increased the risk of liver abnormalities through reciprocal interactions.It could be noted that factors such as insufcient protective facilities, lack of proper warning closures, longer exposure time at risky points, frequent shift work, fatigue, restricted operating and patrolling areas, inadequate surveillance of correct mask types as well as failure to replace masks efectively contributed to increased exposure levels and diminished awareness of selfsafety protection gradually.
Specifcally, liver abnormalities, such as liver calcifcation, multiple cysts, and moderate to severe fatty liver, were considered initial symptoms of nonalcoholic fatty liver disease (NAFLD), as NAFLD encompassed simple steatosis and nonalcoholic steatohepatitis (NASH), which could progress to fbrosis, cirrhosis, and hepatocellular carcinoma (HCC) [31].Excessive accumulation of fat in liver of patients without signifcant alcohol consumption was a major cause of liver dysfunction and chronic liver disease worldwide.In an efort to raise awareness of the disease, the defnition and nomenclature of NAFLD had been changed to metabolic dysfunction-associated fatty liver disease (MAFLD).An international panel of experts from 22 countries had proposed diagnostic criteria for MAFLD, taking into account disease heterogeneity and the role of underlying metabolic factors in disease development.MAFLD was diagnosed in patients with evidence of hepatic steatosis (based on imaging, histopathological examination, or blood biomarker testing) and the presence of at least one of the following three metabolic criteria: obesity/overweight, established type 2 diabetes mellitus (T2DM), or metabolic dysregulation [32].Numerous studies had identifed the association of NAFLD with insulin resistance, metabolic syndrome, diabetes mellitus, and obesity, which led to intrahepatic triglyceride accumulation, increased production of reactive oxygen species, oxidative stress, as well as lipid peroxidation [33].
According to the multiple-hit hypothesis, insulin resistance played a critical role in the pathogenesis of NAFL/ NASH.It led to an increased hepatic de novo lipogenesis and dysfunction of adipose tissue, resulting in elevated levels of circulating free fatty acids.Tese in turn impaired the secretion of adipokines and infammatory cytokines such as IL-6, IL-8, tumor necrosis factor-α (TNF-α) and adiponectin [34] .Based on our results, it appeared that oxidative stress injury caused by VCM exposure was an important mechanism for liver abnormalities during the early screening phase.Abnormal changes in AST/ALT ratio in the blood should be identifed as a specifc indicator for initial screening, especially in conjunction with liver B ultrasonography results.IL-8 and CXCR2 had been shown to contribute to the AST/ALT ratio afection, especially when reached to high levels.Other variables such as longer working years (21 to 30 or 11 to 20), alcohol consumption, shorter sleeping duration (4∼6 hours or less than 4 hours per day), defnite medication history, inadequate protective facilities, and inappropriate mask types were also infuential factors that were to be considered comprehensively.

Conclusions
Tis study made several signifcant fndings.Firstly, it revealed that even with a decrease in VCM concentration among posts in PVC manufacturing factories, liver abnormalities, primarily characterized by fatty liver, liver calcifcation, and liver cysts, could still occur and progressed, due to oxidative stress injury, in which participation of IL-8 and CXCR2 were involved.Secondly, the AST/ALT ratio was regarded as a specifc indicator that could help detecting liver abnormalities before impairment, especially when combined with liver B ultrasonography results.Tird, IL-8 and CXCR2 were noted to correlate strongly with each other and contributed to the alteration of the AST/ALTratio under VCM exposure.Lastly, factors like defnite medication history, inadequate protective facility status (fully broken), alcohol consumption, shorter sleeping duration (4-6 hours and less than 4 hours per day), inappropriate mask types, and longer working years of 21 to 30 (a) or 11 to 20 (a), could contribute to changes in the AST/ALT ratio through complex interactions.Overall, these fndings highlighted the importance on mechanisms underlying liver abnormalities and the signifcance of AST/ALT ratio as an early indicator and the infuence of various variables on liver health.

Limitations
In this study, it is important to acknowledge several limitations that should be considered for future research.Firstly, the sample sizes of the VCM-exposed population were chosen solely from a PVC factory with acetylene hydrochlorination technique, while workers from the ethylene oxychlorination technique were not contained in this study, which would limit the generalization performance to other settings or populations that are afected by VCM.Secondly, results of liver B ultrasonography tests conducted from 2012 to 2018 were categorized as either "normal" or "abnormal" without further detailed analysis on distribution discrepancies and trends of abnormal symptoms among specifc job posts due to data defciency.Tis restricted our ability to perform continuous analysis on these aspects.Tirdly, the distribution and diference analysis of abnormal symptoms among job positions in Group G were compromised due to the unavailability of matched results in 2020.Tis limitation prevented us from fully examining the correlations and interactions between abnormal symptoms and job positions within this specifc group.Fourthly, no such elaborated analysis was taken to conduct the diferential results among 26 Journal of Toxicology groups stratifed through alcohol consumption, such as weekly frequency of alcohol drinking, aging of alcohol consumption, and these regrets will be accomplished in near further.Lastly, the factual causes towards correlation among IL-8, CXCR2, and the AST/ALT ratio were not deeply explored, which will certainly become our critical point in near future, it is important to note that the correlations and interactions observed between IL-8, CXCR2, and other detected indices and variables from questionnaire were only proven on a statistical level.Further assessment is needed to establish their practical relationships with each other.Addressing these limitations in future studies will provide a more comprehensive understanding of the factors infuencing the outcomes observed in this study.

Data Availability
Te original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.Data concerning physical examination indicators, health archives, and occupational history are only available in the occupational physical examination center and the department of occupational health and radiological health, center for disease control and prevention in Bin Hai New Area of Tian Jin City; detection results and processing charts concerning feld investigation as well as layout settings are only available in G and H.However, restrictions apply to the availability of these data and might not be publicly available.

Figure 2 :
Figure2: Tis chart presented signifcant diferences in age distribution (∼30a,∼45a,∼60a) of gender between the two groups.In that, annotation indicated the quantity of people among each age range in either sex, * * indicates P < 0.05.

Figure 4 :
Figure 4: Charts (a-d) demonstrated the 95% CI (upper and lower limits) and OR values for liver B ultrasonography results from the exposed group that could only be diagnosed as normal vs abnormal at 2012 (a), 2014 (b), 2016 (c), and 2018 (d) among health indicators by using a binary logistical regression model ( * * * * P < 0.001, * * P < 0.05).

Figure 7 :
Figure7: Scatter diagrams (a-i) displayed the correlations between VCM concentration and TDGA content among posts of synthetic operators (a), refrigerating operators (b), aggregated operators (c), stripping operators (d), polymerization cleaners (e), maintenance repairers (f ), analytical technicians (g), and feld samplers (h), as well as operators in control group (i), and the r value indicated correlation intensity coefcient.In that, the strong correlations referred to r > 0.7; the moderated correlation referred to 0.4 < r < 0.7; the mild correlation referred to r < 0.4 as * * P < 0.05 and * * * * P < 0.001.Chart (j) presented signifcant diferences between VCM concentration and TDGA content among posts in exposed group and oriented tendencies among them as * * P < 0.05 and * * * * P < 0.001.

Figure 10 :
Figure 10: Scatter charts (a-h) presented the correlation intensities of TDGA content with IL-8, CXCR2, 8-OHdG, and other oxidative injury indices among posts in exposed group.r represented the correlation intensity coefcient (r < 0.4 for mild intensity, 0.4 < r < 0.7 for moderate intensity, r > 0.7 for strong intensity), * * P < 0.05 and * * * * P < 0.001.Chart (a) indicated correlations among indices in synthesis operators; chart (b) indicated correlations among indices in refrigerating operators; chart (c) indicated correlations among indices in aggregated operators; chart (d) indicated correlations among indices in stripping operators; chart (e) indicated correlations among indices in polymerization cleaners; chart (f ) indicated correlations among indices in maintenance repairers; chart (g) indicated correlations among indices in analytical technicians; chart (h) indicated correlations among indices in feld samplers; chart (i) indicated correlations among indices in operators of the control group.
1 mmol/L were included.Moreover, the results from echo-color Doppler ultrasonography tests (referred to as liver B ultrasonography test) conducted in 2012, 2014, 2016, and 2018 were further elaborated in 2020.Te classifcations were expanded to include fatty liver (ranging from moderate

Table 2 :
Multiple logistic regression analysis for liver B ultrasonography results between groups in 2020.

Table 3 :
Multiple linear regression analysis of 8-OHdG content and AST/ALT ratio in 2020.