Occupational cadmium exposure and endocrine biomarker alterations in male workers: a cross-sectional study

Abstract

Background

Occupational exposure to cadmium (Cd) remains an important environmental health concern, particularly in industrial settings where long-term exposure may affect multiple physiological systems. Although Cd has been linked to reproductive and cardiovascular toxicity, human studies that simultaneously evaluate biomarkers of endocrine and endothelial dysfunction remain limited. This cross-sectional study examined the association between occupational Cd exposure and hormonal and endothelial biomarkers in adult male workers.

Methods

This cross-sectional study included 170 men, comprising 85 cadmium-exposed workers and 85 unexposed controls. Urinary cadmium concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS). Serum levels of total testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), arginine, and citrulline were measured using standard laboratory methods. Group comparisons and correlation analyses were conducted to assess associations between cadmium exposure and biomarker profiles.

Results

Cadmium-exposed workers had substantially higher urinary Cd concentrations than controls and higher ADMA and SDMA levels (both p < 0.001). In contrast, total testosterone, FSH, and the arginine/ADMA ratio were significantly lower in the exposed group, while LH showed a smaller but statistically significant reduction. Urinary Cd was inversely correlated with total testosterone and FSH, indicating that a higher internal cadmium burden was associated with less favorable endocrine biomarker profiles.

Conclusions

Occupational cadmium exposure was associated with concurrent changes in endocrine and endothelial biomarkers among male workers. These findings suggest that combined assessment of endocrine and endothelial biomarkers may be useful for occupational monitoring of workers with chronic cadmium exposure.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12902-026-02605-3.

Introduction

Occupational exposure to environmental toxicants remains an important public health concern worldwide, particularly in industrial environments where workers may experience chronic heavy-metal exposure. Among these toxicants, cadmium (Cd) is a persistent, bioaccumulative metal associated with cardiovascular, renal, and reproductive dysfunction. Despite growing recognition of cadmium toxicity, human studies that simultaneously assess alterations in both endocrine and endothelial biomarkers in occupationally exposed populations remain limited [1, 2]. Chronic exposure, primarily through industrial contact or contaminated food and water, has been associated with multisystem toxicity [3]. Although cadmium-related cardiovascular and reproductive effects have both been described, these outcomes are often studied separately, and the extent to which they may coexist within the same exposed population remains insufficiently characterized.

Among the diverse toxicological targets of cadmium, the vascular endothelium has attracted increasing attention because of its role in regulating vascular tone and tissue perfusion. By influencing asymmetric dimethylarginine (ADMA) and endothelial nitric oxide synthase (eNOS)-related pathways, cadmium exposure may compromise nitric oxide bioavailability [4–6]. This is relevant because endothelial dysfunction may affect not only systemic vascular health but also highly vascularized tissues, including the testicular and penile circulation [7, 8]. Accordingly, endothelial injury may contribute to the broader biological effects of cadmium exposure beyond direct cellular toxicity.

Vascular regulation is also closely linked to reproductive physiology. Nitric oxide-mediated vasodilation plays an important role in maintaining testicular blood flow and supporting spermatogenesis and steroidogenesis, which are essential for normal hypothalamic–pituitary–gonadal (HPG) axis function [9–12]. In addition, testosterone has been reported to exert vasoprotective effects, partly through mechanisms involving nitric oxide signaling [13]. These observations suggest that vascular and reproductive alterations may be biologically interconnected. Consistent with this view, previous studies have shown that reproductive toxicity and vascular impairment can both occur in the context of chronic environmental exposure and may together contribute to a broader pattern of systemic dysfunction [14, 15].

Despite the close relationship between vascular and reproductive health, few studies have simultaneously evaluated endothelial and hormonal biomarkers within the same exposed population, particularly among high-risk metallurgical workers. To address this gap, the present study aimed to evaluate markers of endothelial dysfunction (ADMA, SDMA, and the arginine/ADMA ratio) together with reproductive hormone status (total testosterone, FSH, and LH) in adult men occupationally exposed to cadmium.

Methods

Cohort selection

Participants were recruited according to predefined inclusion and exclusion criteria. Individuals with known chronic systemic disease, relevant medication or supplement use, or incomplete biomonitoring and hormonal data were excluded from the final analysis. Initially, 156 cadmium-exposed workers were screened for eligibility. After application of the predefined exclusion criteria, 71 workers were excluded, including individuals with smoking history (n = 23), hypertension (n = 15), diabetes mellitus (n = 9), active vitamin or dietary supplement use (n = 7), renal disease (n = 2), and other exclusion criteria (n = 15). Consequently, 85 cadmium-exposed workers met all eligibility criteria and were included in the final analysis. An equal number of healthy controls (n = 85) were recruited for comparison.

Participants were recruited from two separate facilities operated by the same metallurgical enterprise in Central Anatolia, Türkiye. The cadmium-exposed group consisted of workers involved in cadmium-related metallurgical processes, whereas the control group consisted of administrative staff living and working in the same geographic region. Sociodemographic information collected using the study-specific data collection form included educational attainment, marital status, and place of residence. Individual-level race/ethnicity data were unavailable, and household income was not collected in the original study questionnaire.

Cd-exposed workers

The exposed group consisted of workers employed at two facilities operated by the same metallurgical enterprise. These workers performed physically demanding tasks involving the smelting, refining, and processing of cadmium-containing raw materials, with occupational exposure expected primarily through inhalation of metal fumes and dust generated during these operations. Individual personal or environmental air cadmium monitoring data were not available for all participants in the present analysis. Therefore, occupational cadmium exposure classification was based on job title, documented involvement in cadmium-containing metallurgical processes, occupational history, and urinary cadmium concentrations.

To reduce confounding due to co-exposure, workers with documented significant occupational exposure to other heavy metals, organic solvents, plastics, or other known endothelial or reproductive toxicants were excluded. For exploratory subgroup analyses, the exposed cohort was stratified according to employment duration into short-term exposure (1–5 years) and long-term exposure (≥ 6 years). The 5-year cutoff was selected pragmatically to create two interpretable employment-duration categories with sufficient subgroup sizes for exploratory comparison; it was not intended to represent a biologically validated threshold for cadmium toxicity.

Healthy controls

Healthy controls (n = 85) were recruited from administrative staff who lived and worked in the same geographic area. While this approach was chosen to balance socio-economic and environmental backgrounds broadly, it inherently resulted in a control group with a more sedentary work routine and a different age distribution compared to the physically active factory workers.

They had no known occupational exposure to cadmium, heavy metals, or other harmful industrial chemicals, as assessed by the study-specific data collection form (Supplementary File 1), and their baseline urinary cadmium levels were consistent with background environmental exposure. Background environmental exposure was defined as urinary cadmium concentrations compatible with non-occupational environmental exposure and below health-based human biomonitoring guidance values. In this context, urinary cadmium levels in the control group were interpreted with reference to the German Human Biomonitoring Commission/HBM4EU guidance value for adults, which reports an HBM-I value of 1 µg/L or 1 µg/g creatinine for urinary cadmium. Urinary cadmium was considered an appropriate biomarker because it primarily reflects cumulative cadmium body burden [16, 17].

Exclusion criteria and clinical verification

To minimize major confounding factors affecting endothelial and endocrine function, all participants in both groups were required to be non-smokers. Individuals with a history of hypertension, diabetes mellitus, renal disease, prostate pathology, cardiovascular disease, active infection, or regular medication potentially affecting endocrine or endothelial biomarkers were strictly excluded. Furthermore, participants actively using vitamins, dietary supplements, or essential trace elements, particularly zinc or selenium formulations, were excluded from the study. Among the workers initially screened for eligibility, 7 individuals were excluded because of active use of vitamins, dietary supplements, or trace-element formulations, including zinc- or selenium-containing products. This step was taken to minimize potential nutritional or trace-element-related confounding, as these compounds may influence cadmium kinetics, oxidative stress responses, and endocrine or endothelial biomarker profiles. The absence of these underlying comorbidities was verified using recent routine occupational health screening records, which included fasting blood glucose, kidney function tests (eGFR and serum creatinine), electrocardiograms (ECG), and prostate-specific antigen (PSA) levels. Normal baseline renal parameters (eGFR and creatinine) were specifically confirmed to characterize the study population and to document the absence of overt renal impairment at the time of assessment. Body weight was recorded at the time of sample collection. Data were collected using a study-specific form (Supplementary File 1) to record demographic characteristics, occupational history, clinical exclusion-related information, and laboratory variables.

Ethics approval and consent to participate

All procedures performed in this study involving human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The study protocol was approved by the Medical Research Scientific and Ethical Evaluation Board (TABED) of Ankara Bilkent City Hospital, Ministry of Health, Türkiye (Approval No: TABED-1-24-401; August 28, 2024). Written informed consent was obtained from all participants prior to enrolment.

Sampling and laboratory procedures

Blood and urine samples were collected under controlled pre-analytical conditions. First-void morning urine samples were obtained in sterile polypropylene containers and stored at − 20 °C until analysis. Venous blood was collected into trace-element–free BD Vacutainer^® tubes (Becton Dickinson, USA), centrifuged at 3000 × g for 10 min at 4 °C, and serum aliquots were stored at − 80 °C. Serum total testosterone, LH, FSH, ADMA, SDMA, arginine, and citrulline levels were subsequently analyzed using validated analytical techniques.

Cadmium and creatinine analysis (ICP-MS)

Urinary cadmium concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS). For each sample, 1 mL of urine was digested in closed Teflon vessels using a microwave-assisted procedure with a mixture of 5 mL ultrapure nitric acid (HNO₃) and 5 mL ultrapure deionized water. The digested samples were then diluted to 20 mL with ultrapure water. Analyses were performed using a Thermo Scientific iCAP Qc ICP-MS system (USA) under optimized instrumental conditions to minimize matrix interference. An eight-point external calibration curve ranging from 0.1 to 100 µg/L (r = 0.9999) was used for quantification. The limit of detection (LOD) was 0.0033 µg/L, and procedural blanks measured 0.009 µg/L. All samples and standards were analyzed in triplicate, with relative standard deviation values below 5%. Urinary creatinine concentrations were determined using the Jaffe colorimetric method on an automated clinical chemistry analyzer (Roche Cobas c501, Roche Diagnostics, Mannheim, Germany). Cadmium exposure levels were expressed both as absolute concentrations (µg/L) and as creatinine-corrected concentrations (µg/g creatinine).

Hematological and routine biochemical parameters

Complete blood count (CBC) parameters, including hemoglobin (HGB), hematocrit (HCT), and platelet (PLT) counts, were analyzed using an automated hematology analyzer (Sysmex XN-1000, Sysmex Corporation, Kobe, Japan).

Endothelial biomarkers (ADMA, SDMA, arginine, and citrulline analysis by LC-MS/MS)

The quantification of asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-arginine, and L-citrulline was performed to assess endothelial biomarker profiles. Serum concentrations were determined using a validated LC-MS/MS method under standardized analytical conditions. Arginine and citrulline were included to characterize nitric oxide-related endothelial metabolism better, as arginine serves as the substrate for endothelial nitric oxide synthase (eNOS) and citrulline is generated as a byproduct of nitric oxide synthesis. Because ADMA competitively inhibits eNOS by blocking arginine binding, the arginine/ADMA ratio provides an indirect estimate of functional eNOS activity. Serum ADMA, SDMA, arginine, and citrulline levels were measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS) on a Shimadzu LC-20AD system coupled to an AB Sciex API 3200 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. Chromatographic separation was achieved using a Phenomenex Luna C18 column. For sample preparation, 200 µL of serum was mixed with 100 µL of a deuterated internal standard solution (d7-ADMA) in methanol to precipitate proteins. After centrifugation at 13,000 rpm for 10 min at 4 °C, the supernatant was evaporated under nitrogen at 60 °C. The residue was derivatized with 5% (v/v) acetyl chloride in n-butanol and incubated at 60 °C for 20 min. Following a second evaporation step, samples were reconstituted in a solvent mixture of water, methanol, and formic acid. A 40 µL aliquot was injected into the LC-MS/MS system. Quantification was performed using multiple reaction monitoring (MRM), and the analytical method demonstrated intra-assay and inter-assay coefficients of variation of 2.8% and 3.7%, respectively.

Hormone analysis (ECLIA and ELISA)

Serum total testosterone concentrations were measured using electrochemiluminescence immunoassay (ECLIA) on a Roche Cobas e601 analyser (Roche Diagnostics, Mannheim, Germany). The testosterone measurement range was 0.025–15 ng/mL, with an assay sensitivity of 0.025 ng/mL. Serum LH and FSH concentrations were determined using commercially available sandwich ELISA kits (Human LH ELISA Kit and Human FSH ELISA Kit, Elabscience Biotechnology Inc., USA). Optical density was recorded using a microplate reader (BioTek Synergy HT, BioTek Instruments, Winooski, VT, USA). The assay characteristics were as follows: LH measurement range 0.5–40 IU/L with a sensitivity of 0.1 IU/L; FSH measurement range 0.2–100 IU/L with a sensitivity of 0.1 IU/L.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics version 20.0. Continuous variables were expressed as mean ± standard deviation. Normality of continuous variables was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene’s test. Between-group comparisons were conducted using independent-samples t-tests after assessment of normality and homogeneity of variance. Variables demonstrating non-normality and/or heteroscedasticity were interpreted with caution, and correlation analyses were performed using Spearman’s rank-order correlation.

To account for multiple comparisons across the primary biomarker outcomes, p-values were additionally adjusted using the Benjamini–Hochberg false discovery rate (FDR) procedure, and FDR-adjusted q-values were reported. A two-sided p-value < 0.05 was considered statistically significant for unadjusted analyses, whereas q < 0.05 was considered statistically significant for FDR-adjusted primary biomarker comparisons. Additional adjusted analyses were performed using analysis of covariance (ANCOVA) and multivariable linear regression models. ANCOVA was used to evaluate between-group differences in endocrine biomarkers after adjustment for age. Multivariable linear regression models were constructed to assess whether urinary cadmium concentrations remained associated with key endocrine outcomes after adjustment for available covariates, including age and body weight. Body mass index and waist circumference could not be included because complete data were not available. Exposure-duration subgroup analyses were considered exploratory.

Results

Additional sociodemographic characteristics were evaluated to further characterize the study population. Regarding educational attainment, in the exposed and control groups, respectively, 38 (44.7%) and 38 (44.7%) participants had completed high school, 14 (16.5%) and 13 (15.3%) elementary school, 11 (12.9%) and 12 (14.1%) middle school, 11 (12.9%) and 10 (11.8%) an associate degree, and 11 (12.9%) and 12 (14.1%) a bachelor’s degree.

Regarding marital status, 83 participants (97.6%) in the exposed group were married and 2 (2.4%) were single. In the control group, 79 participants (92.9%) were married, 3 (3.5%) were single, and 3 (3.5%) were widowed. All participants in both groups resided in urban areas in Central Anatolia, Türkiye. Individual-level race/ethnicity data were unavailable, and household income was not collected as part of the original study questionnaire.

Table 1 presents the baseline characteristics and biochemical findings of the study population. Urinary cadmium concentrations, expressed both as absolute values and as creatinine-corrected values, were markedly higher in the cadmium-exposed group than in controls (both p < 0.001). The exposed group was also younger than the control group (p = 0.001).

Table 1.

Baseline characteristics and biochemical parameters in cadmium-exposed and control groups

		N	Mean	Std. Deviation	t	P	q(FDR)
Ages (years)	Cd Exposed	85	40.16	7.72	3.532	0.001	0.001
	Control	85	44.74	9.12
Cd-µg/L (µg/g creatinine)	Cd Exposed	85	3.25 (3.33)	0.69 (0.71)	26.718 (26.647)	< 0.001 (< 0.001)	< 0.001
	Control	85	0.62 (0.64)	0.59 (0.60)
TT (ng/dL)	Cd Exposed	85	494.49	76.04	16.129	< 0.001	< 0.001
	Control	85	662.71	58.85
LH (1.3–9.6 IU/L)	Cd Exposed	85	6.34	1.45	2.235	0.029	0.039
	Control	85	6.95	1.09
FSH (1.2–15.8 IU/L)	Cd Exposed	85	4.13	1.18	14.197	< 0.001	< 0.001
	Control	85	8.36	1.11
HGB (g/dL)	Cd Exposed	85	14.7	0.78	2.354	0.02	0.032
	Control	85	14.96	0.61
HCT (%)	Cd Exposed	85	45.84	2.3	-1.684	0.094	0.105
	Control	85	46.36	1.67
PLT (×10³/µL)	Cd Exposed	85	226.75	40.87	0.474	0.636	0.636
	Control	85	223.98	35.36
Arginine (µmol/L)	Cd Exposed	85	89.72	41.69	-1.383	0.169	0.186
	Control	85	101.71	68.15
Citrulline (µmol/L)	Cd Exposed	85	16.48	10.11	3.146	0.002	0.004
	Control	85	20.27	4.61
ADMA (µmol/L)	Cd Exposed	85	0.27	0.14	3.93	< 0.001	< 0.001
	Control	85	0.22	0.08
SDMA (µmol/L)	Cd Exposed	85	0.24	0.06	4.289	< 0.001	< 0.001
	Control	85	0.21	0.03
Arginine/ADMA	Cd Exposed	85	408.92	249.32	3.003	0.003	0.005
	Control	85	553.21	366.15
SDMA/ADMA	Cd Exposed	85	0.98	0.23	3.973	< 0.001	< 0.001
	Control	85	1.12	0.22

Values are presented as mean ± SD. Differences between the exposed and control groups were assessed using an independent-samples t-test. Urinary cadmium concentrations are reported as absolute values (µg/L) and as creatinine-corrected values (µg/g creatinine). P-values were also adjusted using the Benjamini–Hochberg false discovery rate (FDR) procedure. Adjusted q-values account for multiple comparisons across the primary biomarker outcomes

With respect to endocrine biomarkers, serum total testosterone and FSH levels were significantly lower in cadmium-exposed workers than in controls (both p < 0.001). LH levels were also lower in the exposed group, although the between-group difference was modest in magnitude (p = 0.029). Because the cadmium-exposed group was significantly younger than the control group, age-adjusted ANCOVA analyses were performed for the main endocrine outcomes. After adjustment for age, the between-group differences remained statistically significant for total testosterone (F = 382.61, p < 0.001), FSH (F = 377.96, p < 0.001), and LH (F = 5.85, p = 0.017), indicating that the observed endocrine differences were not attributable solely to age differences between groups.

Regarding endothelial biomarkers, ADMA and SDMA concentrations were significantly higher in the exposed group than in the control group (both p < 0.001). Arginine levels did not differ significantly between groups, whereas citrulline levels were significantly lower in the cadmium-exposed group. In parallel, the arginine/ADMA and SDMA/ADMA ratios were significantly lower among exposed workers, indicating a less favorable endothelial biomarker profile associated with occupational cadmium exposure. To address multiple testing, the Benjamini–Hochberg false discovery rate (FDR) correction was applied to the primary biomarker comparisons presented in Table 1. After FDR adjustment, between-group differences in total testosterone, LH, FSH, hemoglobin, citrulline, ADMA, SDMA, arginine/ADMA ratio, and SDMA/ADMA ratio remained statistically significant, indicating that the principal findings were robust to multiple-comparison correction.

Spearman rank-order correlation analysis showed that urinary cadmium concentrations were inversely associated with total testosterone (ρ = −0.727, p < 0.001), FSH (ρ = −0.738, p < 0.001), LH (ρ = −0.181, p = 0.018), citrulline (ρ = −0.322, p < 0.001), the arginine/ADMA ratio (ρ = −0.201, p = 0.009), and the SDMA/ADMA ratio (ρ = −0.359, p < 0.001). Positive correlations were observed between urinary cadmium and ADMA (ρ = 0.321, p < 0.001) and SDMA (ρ = 0.207, p = 0.007). Overall, the Spearman correlation matrix indicated that higher urinary cadmium concentrations were associated with lower endocrine biomarker levels and a less favorable endothelial biomarker profile, as illustrated in Fig. 1.

Fig. 1.

Spearman correlation heatmap of urinary cadmium, endocrine biomarkers, and endothelial biomarkers

Figure 2 further illustrates the inverse relationship between urinary cadmium and total testosterone, showing a clear negative linear trend (R² = 0.602). Overall, the correlation matrix suggests that a higher internal cadmium burden was associated with less favorable endocrine and endothelial biomarker profiles.

Fig. 2.

Scatter plot of the association between urinary cadmium and total testosterone levels

The heatmap shows Spearman rank-order correlation coefficients among urinary cadmium concentrations, reproductive hormones (total testosterone, LH, and FSH), and endothelial biomarkers (ADMA, SDMA, and the arginine/ADMA ratio). Negative coefficients indicate inverse associations, whereas positive coefficients indicate direct associations between variables. Color intensity reflects the strength and direction of the correlation coefficients.

Each point represents an individual participant. The fitted regression line shows an inverse association between urinary cadmium concentrations and serum total testosterone levels, indicating that a higher internal cadmium burden was associated with lower testosterone concentrations.

To assess whether biomarker patterns differed by employment duration, cadmium-exposed workers were stratified into short-term (1–5 years) and long-term (≥ 6 years) exposure groups (Table 2). Workers with longer exposure durations had significantly higher urinary cadmium concentrations than those with shorter exposure durations, both in absolute terms and after creatinine correction (p = 0.011 and p = 0.014, respectively).

Table 2.

Biomarker and selected clinical parameters according to exposure duration among cadmium-exposed workers

	Employment Duration Category	N	Mean	Std. Deviation	t	p
Ages (years)	Short duration (1–5 years)	32	38.91	7.40	-1.171	0.245
	Long duration (≥ 6 years)	53	40.92	7.87
Weight (kg)	Short duration (1–5 years)	32	82.53	8.09	− 0.132	0.895
	Long duration (≥ 6 years)	53	82.75	7.20
Cd-µg/L (µg/g creatinine)	Short duration (1–5 years)	32	3.01 (3.11)	0.73 (0.82)	-2.615 (-2.627)	0.011 (0.014)
	Long duration (≥ 6 years)	53	3.40 (3.48)	0.63 (0.68)
TT (ng/dL)	Short duration (1–5 years)	32	529.31	67.25	3.492	0.001
	Long duration (≥ 6 years)	53	473.47	73.80
LH (1.3–9.6 IU/L)	Short duration (1–5 years)	32	6.15	1.65	− 0.816	0.417
	Long duration (≥ 6 years)	53	6.45	1.64
FSH (1.2–15.8 IU/L)	Short duration (1–5 years)	32	4.08	1.09	− 0.266	0.791
	Long duration (≥ 6 years)	53	4.16	1.46
HGB (g/dL)	Short duration (1–5 years)	32	14.67	0.72	− 0.306	0.761
	Long duration (≥ 6 years)	53	14.72	0.82
HCT (%)	Short duration (1–5 years)	32	45.79	2.24	− 0.159	0.874
	Long duration (≥ 6 years)	53	45.87	2.36
PLT (×10³/µL)	Short duration (1–5 years)	32	235.94	39.84	1.625	0.108
	Long duration (≥ 6 years)	53	221.21	40.86
Arginine (µmol/L)	Short duration (1–5 years)	32	84.16	40.38	− 0.955	0.342
	Long duration (≥ 6 years)	53	93.08	42.48
Citrulline (µmol/L)	Short duration (1–5 years)	32	15.91	8.78	− 0.405	0.686
	Long duration (≥ 6 years)	53	16.83	10.91
ADMA (µmol/L)	Short duration (1–5 years)	32	0.22	0.06	-2.423	0.018
	Long duration (≥ 6 years)	53	0.29	0.16
SDMA (µmol/L)	Short duration (1–5 years)	32	0.22	0.05	-1.714	0.090
	Long duration (≥ 6 years)	53	0.25	0.06
Arginine/ADMA	Short duration (1–5 years)	32	401.38	205.32	− 0.215	0.830
	Long duration (≥ 6 years)	53	413.47	274.26
SDMA/ADMA	Short duration (1–5 years)	32	1.04	0.17	1.640	0.105
	Long duration (≥ 6 years)	53	0.95	0.25

Values are presented as mean ± SD. Comparisons between short-term (1–5 years) and long-term (≥ 6 years) exposure groups were performed using the independent-samples t-test. Urinary cadmium concentrations are reported both as absolute values (µg/L) and as creatinine-corrected values (µg/g creatinine)

Long-term exposed workers also had significantly lower total testosterone levels (p = 0.001) and higher ADMA levels (p = 0.018) than short-term exposed workers. By contrast, LH, FSH, SDMA, arginine/ADMA ratio, SDMA/ADMA ratio, and hematological parameters did not differ significantly between exposure-duration groups.

These exploratory subgroup findings indicate that workers in the longer-duration employment group had higher urinary cadmium concentrations, lower total testosterone levels, and higher ADMA levels. However, these findings should not be interpreted as evidence that exposure duration was independently associated with worse biomarker profiles, because the longer-duration group also had higher internal cadmium burden.

Because the cadmium-exposed group was significantly younger than the control group, additional multivariable analyses were performed. Linear regression models including urinary cadmium concentration, age, and body weight demonstrated that urinary cadmium remained independently associated with lower total testosterone concentrations (B = − 62.61, 95% CI: −69.33 to − 55.89, p < 0.001) and lower FSH concentrations (B = − 1.33, 95% CI: −1.50 to − 1.16, p < 0.001). Age was independently associated with testosterone concentrations but not with FSH, whereas body weight was not significantly associated with either biomarker.

Because exact employment duration in years was not available for all participants, exposure duration could not be analyzed as a true continuous variable. Therefore, exposure-duration category was evaluated as an ordinal predictor in additional adjusted models. Additional age- and body weight-adjusted regression analyses among cadmium-exposed workers showed that the longer exposure-duration category was significantly associated with higher urinary cadmium concentrations (B = 0.38, 95% CI: 0.08 to 0.69, p = 0.014), lower total testosterone concentrations (B = − 50.19, 95% CI: −81.18 to − 19.20, p = 0.002), and higher ADMA levels (B = 0.072, 95% CI: 0.011 to 0.132, p = 0.021). No significant adjusted associations were observed for FSH, LH, SDMA, citrulline, or the arginine/ADMA ratio. Overall, employment-duration category should therefore be interpreted as an approximate proxy for cumulative cadmium burden rather than as an independent biological determinant.

Discussion

The present study showed that occupational cadmium exposure was associated with concurrent changes in endocrine and endothelial biomarkers. Specifically, cadmium-exposed workers had lower total testosterone and FSH levels, higher ADMA and SDMA concentrations, and a lower arginine/ADMA ratio than unexposed controls. These findings indicate that chronic occupational exposure to cadmium may be associated with alterations across multiple physiological systems, including hormonal regulation and endothelial homeostasis. Correlation analyses further support this interpretation, showing that higher urinary cadmium concentrations were associated with less favorable profiles of endocrine and endothelial biomarkers. Importantly, the major findings remained statistically significant after false discovery rate correction, supporting the robustness of the observed alterations in endocrine and endothelial biomarkers.

A key finding of the study was an uneven pattern of endocrine changes across components of the hypothalamic-pituitary-gonadal (HPG) axis. While total testosterone and FSH levels were significantly lower in the exposed group, LH showed only a modest reduction. This hormonal profile is particularly interesting when considered in the context of the participants’ ages. Physiologically, testosterone levels peak around age 30 and gradually decline, whereas FSH and LH levels typically rise in aging men due to reduced negative feedback. Although our exposed group was significantly younger than the control group, it exhibited significantly lower total testosterone and FSH levels. This paradox suggests that the observed hormonal differences are unlikely to be explained solely by the age difference between groups and that they remained independently associated with cadmium exposure in adjusted analyses. The simultaneous reduction of testosterone and FSH may be consistent with alterations involving central components of the HPG axis; however, GnRH secretion and pituitary function were not directly assessed in the present study. Because the present study was cross-sectional and did not include direct functional assessments of the HPG axis, these findings should be interpreted with caution. Nonetheless, the observed hormonal profile is consistent with the possibility of alterations in HPG-axis regulation and testicular steroidogenesis associated with chronic occupational cadmium exposure.

In addition to the endocrine findings, cadmium exposure was linked to a less favorable endothelial biomarker profile. The rise in ADMA and SDMA indicates an adverse shift in pathways related to nitric oxide availability. Because ADMA is an endogenous inhibitor of endothelial nitric oxide synthase, higher ADMA levels are consistent with a less favorable endothelial biomarker profile in workers with chronic exposure. Furthermore, we observed significantly lower arginine/ADMA and SDMA/ADMA ratios in the exposed group. The arginine/ADMA ratio reflects nitric oxide bioavailability; its reduction is consistent with a less favorable endothelial biomarker profile. Regarding routine hematological parameters, HGB showed a small but statistically significant difference between groups, whereas HCT and PLT did not differ significantly. Because these values remained within clinically normal ranges, these findings should be interpreted cautiously and primarily as supportive descriptive data rather than as evidence of clinically meaningful hematological impairment.

The subgroup analysis by employment duration showed that workers with long-term exposure had significantly higher ADMA levels than those with short-term exposure, consistent with an association between longer employment duration and a less favorable endothelial biomarker profile. However, interpreting this exposure-duration subgroup analysis warrants caution. Given the long biological half-life of cadmium (approximately 10–30 years), the physiological difference between 1 and 5 years and ≥ 6 years of exposure might be relatively limited. The less favorable biomarker profile in the long-term exposure group may partly reflect the higher internal cadmium burden observed in this group. Therefore, employment-duration category should be regarded as an approximate proxy for cumulative exposure rather than as evidence of an independent duration effect.

Notably, the correlation pattern observed in this study suggests that endocrine changes and variations in endothelial biomarkers may occur simultaneously among workers exposed to cadmium. However, the current data do not provide sufficient evidence to definitively determine whether these changes arise from a single mechanism or from partially distinct biological processes triggered by the same exposure. Rather than supporting a clear sequential or causal model, our findings suggest that cadmium exposure is associated with parallel alterations in biomarkers across multiple physiological systems. This distinction is important in occupational health because relying on a single biomarker might underestimate the broader biological effects of long-term cadmium exposure. Therefore, integrated biomonitoring approaches could offer a more comprehensive understanding of cadmium-related health risks.

Population-based human studies have reported associations between cadmium exposure and testosterone levels, although the direction and magnitude of these associations have varied across populations and exposure settings [18].

Such discrepancies may reflect differences in exposure levels and durations, biomarker matrices, smoking status, age distributions, dietary factors, and other population-specific characteristics. In contrast, mechanistic and experimental studies have more consistently documented adverse effects of cadmium on the male reproductive system. These varied observations underscore the importance of assessing both hormonal and endothelial parameters within the same study design.

Cadmium exposure has been shown to adversely affect male reproductive tissues, including seminiferous tubules, Sertoli cells, Leydig cells, and the blood–testis barrier [1, 2, 19–21], while broader toxicological evidence indicates that cadmium-induced injury is strongly linked to oxidative stress, redox imbalance, and disruption of cellular defense mechanisms [22]. In individuals with chronic cadmium exposure, endothelial dysfunction has been associated with lower nitrite and L-arginine levels, increased ADMA and soluble thrombomodulin levels, elevated malondialdehyde and protein carbonyl concentrations, and decreased erythrocytic glutathione [23]. These findings support the interpretation that chronic cadmium exposure may impair nitric oxide-related endothelial pathways and promote oxidative vascular injury.

A recent mouse study showed that cadmium exposure reduces testicular weight, markedly impairs Leydig cell function, and dose-dependently decreases 3β-HSD activity. At the same time, endothelial and pericyte markers, including α-SMA, CD31, caveolin-1, and NG2, were significantly elevated, indicating testicular vascular disruption [19]. The reduction in testosterone and the decreased arginine/ADMA ratio observed in our study align with these vascular and hormonal disturbances.

The endocrine-disrupting effects observed here are not limited to cadmium. Elevated levels of cadmium, arsenic, and other heavy metals have also been reported in men with idiopathic hypogonadotropic hypogonadism, with arsenic showing a particularly strong negative correlation with serum testosterone [24]. Similarly, experimental and review evidence indicates that cadmium exposure may impair male reproductive function through testicular degeneration, androgen deprivation, impaired spermatogenesis, hormonal imbalance, and oxidative stress-related testicular injury [20, 21, 25]. Conversely, the lower testosterone and FSH levels, along with the more modest reduction in LH observed in our occupationally exposed cohort, may reflect a pattern associated with chronic human occupational exposure rather than acute experimental toxicity alone.

In line with this, our negative correlation between urinary cadmium and total testosterone is consistent with previous evidence suggesting that cadmium may act as an endocrine-disrupting agent that affects HPG axis function and testicular integrity [20].

Beyond endocrine disruption, recent experimental work summarized by Antar et al. (2024) indicates that cadmium-induced mitochondrial dysfunction, inflammation, and oxidative stress may contribute to both endothelial and testicular injury [21].

All participants also had normal baseline clinical parameters, including eGFR and serum creatinine. Significant endocrine and endothelial biomarker alterations despite normal baseline renal parameters suggest that these changes may be detectable in individuals without overt renal impairment; however, the cross-sectional design does not allow establishment of their temporal sequence. Disruption of these processes may reduce cholesterol transport and impair steroid hormone production. In addition, oxidative stress-related cellular injury, including lipid peroxidation and mitochondrial dysfunction, may further compromise steroidogenic capacity [4, 22]. Overall, the present findings suggest that cadmium exposure may be associated with concurrent changes in hormonal and vascular-related biomarkers in exposed workers.

Limitations

Our study has several important limitations. The cross-sectional design precludes definitive causal inferences about the temporal sequence of events. Therefore, the observed changes in endocrine and endothelial biomarkers should be interpreted as associations rather than as evidence of causation. Longitudinal studies are needed to determine whether cadmium exposure precedes and contributes to these biomarker changes over time.

The study population was highly selected. To minimize major clinical heterogeneity in the assessment of endocrine and endothelial biomarkers, individuals with a history of smoking, hypertension, diabetes mellitus, renal disease, cardiovascular disease, prostate pathology, active infection, use of relevant medications, or active use of vitamins and dietary supplements were excluded. Although this approach helped minimize important biomarker-related sources of variability, it may have reduced external validity and introduced selection bias. Excluding individuals with chronic conditions potentially related to cadmium exposure may have resulted in a healthier exposed cohort and therefore may have led to an underestimation of the true cumulative health effects of occupational cadmium exposure.

Strict individual matching was not performed between the exposed and control groups. The control group consisted of administrative staff who were significantly older and likely more sedentary than the physically active factory workers. Although additional adjusted analyses were performed to account for age and body weight, residual confounding related to age distribution, occupational physical activity, lifestyle characteristics, and unmeasured socioeconomic factors cannot be completely excluded.

The study focused primarily on subclinical biochemical and biomarker alterations and did not systematically evaluate overt clinical manifestations of cadmium toxicity. Functional reproductive outcomes, reproductive symptoms, erectile function, fertility history, and semen parameters were not assessed. Despite our focus on male reproductive toxicity and the observed reductions in total testosterone and FSH, semen analysis could not be performed due to logistical and ethical constraints in the occupational screening context. Therefore, the present findings cannot be directly translated into conclusions about fertility status or clinically evident reproductive dysfunction.

The endocrine assessment was limited to total testosterone, LH, and FSH. Sex hormone-binding globulin (SHBG) was not measured, precluding calculation of biologically active free testosterone. Inhibin B was also not assessed, limiting our ability to evaluate Sertoli cell function and the feedback regulation of FSH. These missing parameters are particularly relevant because the observed hormonal pattern, characterized by reduced total testosterone and FSH with only a modest reduction in LH, may reflect a non-classical or mixed disruption of the hypothalamic–pituitary–gonadal axis that cannot be fully characterized using the available hormonal markers alone.

Anthropometric characterization was limited. Waist circumference was not collected as part of the occupational health screening protocol, and complete height measurements were unavailable for all participants; therefore, body mass index (BMI) could not be reliably calculated for the entire study population. Body weight was the only anthropometric variable available for all participants and was therefore used in the additional adjusted analyses. Nevertheless, residual confounding related to body composition, adiposity, metabolic status, and cardiovascular risk profile cannot be fully excluded.

Serum or urinary trace-element levels, particularly zinc and selenium, were not measured. Although individuals actively taking zinc, selenium, multivitamins, or other dietary supplements were excluded to reduce supplementation-related heterogeneity, the lack of direct trace-element measurements remains an important limitation. This matters because zinc and selenium may influence cadmium kinetics, metallothionein binding, oxidative stress responses, and endocrine or endothelial biomarker profiles.

Exposure assessment was based primarily on occupational history, job title, documented involvement in cadmium-containing metallurgical processes, and urinary cadmium concentrations. Individual personal or environmental air cadmium monitoring data were not available for all participants in this analysis. Although urinary cadmium is an established biomarker of cumulative internal cadmium burden, the lack of comprehensive individual-level air-monitoring data limited our ability to directly quantify the intensity of external workplace exposure.

Finally, the exposure-duration subgroup analyses should be interpreted as exploratory. The relatively small subgroup sizes also limited statistical power, and these analyses should therefore be regarded as hypothesis generating rather than confirmatory. The 5-year cutoff was selected pragmatically to create two interpretable occupational-duration categories with sufficient subgroup sizes and was not intended to represent a biologically validated threshold for cadmium toxicity. Moreover, exact employment duration in years was not available for all participants, precluding analysis of exposure duration as a continuous variable. Because cadmium has a prolonged biological half-life and accumulates over decades, the employment-duration category should be considered an approximate proxy for cumulative exposure rather than an independent biological determinant.

Conclusion

In summary, occupational cadmium exposure was associated with concurrent alterations in endocrine and endothelial biomarkers in male workers. These alterations included lower total testosterone and FSH levels, a slight decrease in LH, and a less favorable endothelial biomarker profile characterized by higher ADMA and SDMA levels and a reduced arginine/ADMA ratio. Collectively, these findings indicate that long-term cadmium exposure is accompanied by simultaneous biomarker shifts across multiple physiological systems. Crucially, given the cross-sectional design of this study, our data only demonstrate the coexistence of these endocrine and endothelial disturbances within the same exposed population. We explicitly acknowledge that coexistence does not establish a causal relationship, and longitudinal studies are required to determine the definitive temporal sequence of these events. Nevertheless, from an occupational health perspective, these findings emphasize the importance of comprehensive biomarker monitoring, as assessing endocrine or endothelial parameters in isolation may underestimate the wider systemic health risks associated with chronic cadmium exposure in industrial workers.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ADMA

Asymmetric dimethylarginine

CBC

Complete blood count

Cd

Cadmium

eGFR

Estimated glomerular filtration rate

eNOS

Endothelial nitric oxide synthase

FSH

Follicle-stimulating hormone

HCT

Haematocrit

HGB

Haemoglobin

HPG

Hypothalamic-pituitary-gonadal

ICP-MS

Inductively coupled plasma mass spectrometry

LC-MS/MS

Liquid chromatography-tandem mass spectrometry

LH

Luteinising hormone

NO

Nitric oxide

PLT

Platelet count

PSA

Prostate-specific antigen

SDMA

Symmetric dimethylarginine

TT

Total testosterone

Author contributions

DOV conceived and designed the study, supervised the research process, interpreted the data, and drafted the manuscript. LT coordinated fieldwork and contributed to exposure assessment and biological sample collection. SBİ oversaw laboratory procedures and contributed to toxicological evaluation and quality control. MGG performed the statistical analyses and contributed to data interpretation. GA conducted biochemical assays, processed samples, and curated data. SD contributed to the clinical interpretation of endocrine parameters and manuscript revision. VAT assisted with literature review, data management, and manuscript editing. ET provided senior supervision, contributed to methodological refinement, and critically reviewed the final manuscript. All authors read and approved the final manuscript.

Funding

The authors declare that no funds, grants, or other financial support were received for the conduct, preparation, or publication of this study.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All procedures performed in this study involving human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The study protocol was approved by the Medical Research Scientific and Ethical Evaluation Board (TABED) of Ankara Bilkent City Hospital, Ministry of Health, Türkiye (Approval No: TABED-1-24-401; August 28, 2024). aWritten informed consent was obtained from all participants prior to enrolment.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.