Biochemical study of the effect of lead exposure in nonobese gasoline station workers and risk of hyperglycemia: A retrospective case-control study

Abstract

Evaluate the relationship between blood lead (Pb) levels and other biomedical markers and the risk of diabetes in gasoline station workers. The participants were separated into 2 groups: group A consisted of 26 workers from gasoline filling stations, while group B comprised 26 healthy individuals. Serum levels of malondialdehyde, IL-1β, visfatin, insulin, fasting blood sugar, and vitamin D were assessed. Mean Pb level was significantly higher in group A compared to group B (almost 2.9 times higher levels) (14.43 ± 1.01 vs 5.01 ± 1.41, µg/dL). The levels of visfatin (23.19 ± 0.96 vs 3.88 ± 0.58, ng/mL), insulin (22.14 ± 1.31 vs 11.26 ± 0.75, mU/L), fasting blood sugar (118.4 ± 26.1 vs 82.7 ± 9.2, gm/dL), malondialdehyde (6.40 ± 0.27 vs 1.62 ± 0.21, nmol/mL), and IL-1β (330.25 ± 10.34 vs 12.35 ± 1.43, pg/mL) were significantly higher in group A, meanwhile; vitamin D (11.99 ± 1.55 vs 35.41 ± 3.16, ng/mL) were significantly lower in group A. A positive association exists between blood Pb levels and increased inflammatory markers. Lead exposure increases serum insulin and fasting blood sugar, which suggests that it is diabetogenic and that increased inflammation is a possible cause.

1. Introduction

Pollution by heavy metals has become one of the world’s most critical environmental problems. Unlike organic pollutants, toxic elements like lead (Pb), mercury, cadmium, and copper are not converted to harmless small molecules by biological interactions.^[1] Lead is found in the Earth’s crust with a bluish-white color and a dazzling luster and crystallizes in a face-centered-cubic structure with unknown allotropic modifications.^[2,3] Pb pollution in the water, air, and agricultural land is a major environmental problem because of its bad effects on human health and the environment.^[4,5] Lead poisoning is a global problem since it can enter the body by inhalation, ingestion, and skin absorption, with inhalation causing the body to absorb larger levels of Pb.^[6] Pb is a neurotoxin that builds up in different organs and tissues, causing neurological problems like behavioral disorders and brain damage, as well as harming general health, cardiovascular, and renal systems.^[7]

Tetraethyllead is a fuel additive that was added for the first time with gasoline as a proprietary octane rating enhancer that allowed engine compression to be increased significantly^[8]; leaded gasoline is considered a severe public health risk since it can induce poisoning after prolonged exposure, particularly in gas station workers. Reactive oxygen species (ROS) have been observed to be generated by Pb.^[9] Excess free radicals like hydroxyl radical, superoxide anion, singlet oxygen, and hydrogen peroxide can oxidize a variety of biomacromolecules, such as unsaturated fatty acids, proteins, and pigments, resulting in membrane damage, inactivation of enzymes, and DNA damage, that is thought to be the primary cause of cell death.^[10,11]

Glutathione (GSH) is a tripeptide that is a crucial cellular antioxidant. After synthesis, GSH can potentially undergo additional metabolism via many pathways.^[12,13] The primary consequence of oxidative stress is the oxidation of GSH to form dimeric glutathione disulfide (GSSG). This transformation is facilitated by the selenoenzyme glutathione peroxidase, which utilizes reducing equivalents from the thiol groups of GSH to stabilize ROS. Subsequently, GSH incorporates a second oxidized GSH molecule to generate the dimeric form of oxidized glutathione, GSSG.^[14] The measurement of intracellular amounts of GSH and GSSG in the bloodstream serves as quantifiable biomarkers for assessing the overall oxidative status of the body in response to oxidative events.^[15] In a typical cellular environment, the reduced form of GSH constitutes a minimum of 90% of the total glutathione content, while the oxidized form of GSSG accounts for <10%. Any alteration in these proportions, characterized by a decrease in GSH and/or an increase in GSSG, may indicate oxidative stress.^[16]

Uncontrolled oxidative stress, which refers to an imbalance between prooxidant and antioxidant levels favoring prooxidants, can result in cellular, tissue, and organ harm due to oxidative damage. The detrimental effects of elevated free radicals or ROS on lipids have been widely acknowledged. The main sources of internally generated ROS are the mitochondria, plasma membrane, endoplasmic reticulum, and peroxisomes.^[17] Lipid peroxidation, the process of oxygen reacting with unsaturated lipids, forms a diverse range of oxidation products. Lipid peroxidation mostly yields lipid hydroperoxides. Malondialdehyde (MDA) is one of the several aldehydes that can be produced as byproducts during lipid peroxidation^[17]; MDA is an extremely mutagenic byproduct of lipid peroxidation.^[17] The enzymatic generation of MDA is a well-established process. However, its biological functions and potential dual role, which may vary depending on the dosage, have not been well investigated. It is worth noting that MDA is more chemically stable and may easily pass through cell membranes compared to ROS.^[18] MDA stimulated the expression of collagen genes in hepatic stellate cells by increasing the expression of the specificity protein-1 gene and the levels of specificity protein-1 and specificity protein-3 proteins.^[19] However, the production of MDA through nonenzymatic processes is poorly understood, even though it has potential therapeutic benefits; this is because MDA is thought to be generated during stressful circumstances and has a strong ability to react with various biomolecules, such as proteins or DNA, resulting in the formation of adducts.^[20,21] Several studies linked exposure to Pb with increased levels of MDA.^[22–24]

Visfatin exhibits a significant presence in visceral adipose tissue, and there is a positive association between visfatin levels in the bloodstream and obesity.^[25] Furthermore, visfatin is recognized as a nicotinamide phosphoribosyl transferase that plays a direct role in the production of nicotinamide adenine dinucleotide.^[26] Multiple lines of evidence indicate that increased visfatin levels are associated with the pro-inflammatory response observed in obesity.^[27] Visfatin was discovered to be mostly secreted by macrophages rather than adipocytes in visceral adipose tissue. Plenty of proof supports the notion that visfatin is expressed by the macrophages that invade adipose tissue and is created as a response to inflammatory signals.^[28,29] Visfatin activities are now understood to have endocrine, paracrine, and autocrine effects. The autocrine activities of visfatin may significantly impact the regulation of insulin sensitivity in the liver.^[30] Research has documented diverse effects and associations between visfatin plasma levels and various medical disorders. It exhibits antiapoptotic properties on neutrophils in animal and clinical sepsis models.^[31] It is also elevated in cases of acute lung injury, serving as a valuable indicator of this condition.^[32] Visfatin levels are lower in patients with steatohepatitis than those with pure steatosis^[33]; nevertheless, there was a favorable correlation between elevated visfatin levels and portal inflammation.^[34] These data indicate a potential correlation between visfatin and inflammation. A study has shown a negative relationship between visfatin levels and creatinine clearance and a positive relationship between visfatin levels and urine albumin excretion; this suggests that the visfatin levels in the bloodstream are affected by kidney function.^[35] Our previous study demonstrated that serum visfatin levels may play a crucial role in regulating obesity and osteoarthritis and can be a significant factor when combined with excessive environmental exposure to Pb in developing these conditions.^[36]

Vitamin D has been associated with various physiological processes beyond its traditional impact on bone mineralization.^[37] Due to advancements in building constructions, transportation, and industry, there has been an increase in the global prevalence of vitamin D deficiency (VDD) and Pb poisoning. These issues pose major challenges, particularly among children.^[38–40] There is a suggestion that exposure to high Pb levels is a potential risk factor for VDD.^[41,42] Research on children with high levels of Pb exposure has shown a clear and strong link between the concentration of 1,25-dihydroxyvitamin D [1,25(OH)2D] and blood Pb levels.^[43,44] Elevated blood Pb levels have been proposed to interfere with the renal conversion of 25-hydroxyvitamin D [25(OH)D] to its active form, 1,25(OH)2D, by the action of 1-α-hydroxylase.^[45] As a result, the decrease in the production of serum 1,25(OH)2D caused by Pb toxicity is accompanied by lower levels of calcium and higher levels of parathyroid hormone in the blood. Therefore, Pb disrupts multiple vitamin D actions essential for maintaining calcium homeostasis and regulating metabolism in different tissues and organs.^[46,47]

Interleukin (IL)-1β, a cytokine that promotes inflammation, has been identified as a significant cause of damage to β-cells. Macrophages in β-cells are the primary sources of IL-1β production. The levels of IL-1β, a powerful cytokine that promotes inflammation, are tightly controlled by IL-1 receptor antagonist (IL-1Ra). Macrophages are the primary producers of IL-1β/IL-1Ra during inflammation, creating both cytokines in an automated feedback loop.^[48,49] Beta cells exhibit a higher IL-1 receptor (IL-1R) level than other cells. Therefore, maintaining an appropriate equilibrium between IL-1β and IL-1Ra levels is essential in determining the reaction of β-cells and, consequently, the advancement of type 2 diabetes mellitus (DM).^[48] IL-1β has multiple roles in controlling inflammatory responses and metabolism. It can regulate insulin release and stimulate the death of β cells, ultimately developing type 2 diabetes mellitus.^[50,51] Signaling events of IL-1β trigger an acute phase response, hypotension, vasodilation, and pyrexia, ultimately leading to significant inflammatory events.^[52] Prolonged exposure to Pb and cadmium in humans notably affected IL-1β.^[53] Lead has a detrimental impact on the metabolism of cytokines, specifically ILs IL-2, IL-1β, IL-6, IL-4, IL-8. It also affects the expression and functioning of inflammatory enzymes such as cyclooxygenases.^[54,55] The levels of IL-1β and IL-6 in the progeny of mice exposed to Pb were elevated. The elevated levels of IL-1β and IL-6 in the hippocampus of offspring may contribute to the neurotoxic effects linked to maternal exposure to Pb.^[56]

We undertook this study to investigate the possible risk of DM with environmental exposure to Pb and its biochemical pathways. This study aimed to investigate the potential association between blood Pb levels and other biological markers, including MDA, IL-1β, visfatin, insulin, fasting blood sugar (FBS), and vitamin D, and the possible risk of DM among a cohort of gasoline station workers in Iraq.

2. Materials and methods

2.1. Study design and study population

The details of the study sample were acquired through the administration of a questionnaire interview. This study involved recruiting 52 males who were not obese and had an age range of 25 to 50 years. These participants were separated into 2 groups: group A consisted of 26 workers from gasoline filling stations, while group B comprised 26 healthy individuals without any health problems or chronic diseases.

All the exposed workers had been employed in their present roles for at least 1 year. Every participant had an interview process to gather information about their overall health, lifestyle choices, smoking behaviors, and past instances of exposure. Income was based on the United Nations Population Fund classification of Iraq into 4 monthly income categories (using Iraqi Dinar [ID])^[57]: poor (<500,000 ID), low (500,000–749,000 ID), middle (750,000–999,000 ID), and high (≥1,000,000 ID).

Blood samples were collected from all participants to measure the concentrations of Pb, visfatin, IL-1β, insulin, vitamin D, and MDA. The atomic absorption spectrometric approach detected Pb levels in blood samples.^[58]

2.2. Study settings

The study was carried out in Baghdad governorate, Iraq. The study was carried out in 5 gasoline stations (these stations supply leaded gasoline), which were randomly selected from 117 gasoline stations in Baghdad (stations names: Al-Wefaq, Musa Bin Naseer, Al-Saadoun, Al-Jauadian, and Al-Kilani, respectively). The participants were chosen by a nonrandom quota sampling method, and their distribution was based on their region of residency, as determined by the statistics provided by the agriculture organization in Baghdad. The average weekly work hours were 81.4 ± 6.4 hours, and most workers have been working for at least 1 year. The study was conducted from February 1, 2022 to July 1, 2022.

2.3. Inclusion criteria

1. Male sex.

2. Work for at least 1 year.

3. The rationale behind selecting controls (group B) was their alignment with the study respondents (group A) regarding residence and socioeconomic backgrounds.

4. All participants were interviewed to assess potential exposure to Pb, and none reported any such exposure (other than their current occupation).

2.4. Exclusion criteria

1. Female sex.

2. Past or current exposure to Pb.

3. The control group participants lived without any nearby factories or fuel stations.

2.5. Laboratory analysis

The authors were responsible for the collection of the blood samples. A satisfactory whole blood sample was acquired by a skin-puncture technique referred to as a finger stick, following a comprehensive hand-washing procedure to prevent any potential contamination. The lateral aspect of the third digit was utilized. A specialized capillary tube (Bioevopeak^®, Shandong, China) containing heparin to prevent coagulation was employed to precisely collect a 50 μL volume of whole blood. In every experiment, a precise volume of 50 μL of whole blood was transferred from the capillary tube to the treatment reagent tube using specialized plungers. About 2.5 mL of blood was dispensed into a plastic tube containing ethylenediaminetetraacetic acid to estimate Pb.

2.5.1. Measurement of Pb (µg/dL)

A volume of 2.5 mL of whole blood was thoroughly mixed with an equal volume of trichloroacetic acid using a wooden stick. The resulting mixture was centrifuged at a speed of 3000 revolutions per minute for 10 minutes (Thermos Scientific^®, Greenville) to eliminate cellular debris. The supernatant was carefully transferred to a sterile tube and afterward aspirated straight into the Atomic Absorption Spectrophotometer (Perkin Elmer model 303 graphite furnace).^[59–61]

2.5.2. Measurement of biomarkers

The serum samples were used to determine the level of MDA, nmol/mL (MDA ELISA Kit, product ID E0048Ge; Sunlong Biotech^®, HangZhou, China), Visfatin, ng/mL (Human Visfatin ELISA Kit, product ID SL1825Hu; Sunlong Biotech^®), IL-1β, pg/mL (Human Interleukin 1-β ELISA Kit, product ID SL0984Hu; Sunlong Biotech^®), Insulin, mU/L (Human Insulin ELISA Kit, product ID SL0933Hu; Sunlong Biotech^®), and Vitamin D, ng/mL (Human Vitamin D3, VD3 ELISA kit, product ID SL1833Hu; Sunlong Biotech^®), by ELISA technique by manufacturer’s procedure.

In brief, 50 µL of serum samples were put in wells of ELISA plates for 2 hours at room temperature. Then, 50 µL of detection antibody was added for 90 minutes.^[62] It’s followed by washing 3 times using a prepared washing buffer. The spectrophotometer was used to measure the optical density of samples, and a standard curve was used to assess the concentration of samples (ELISA reader; Diagnostic Automation/Cortez Diagnostics^®, Calabasas).

2.5.3. Measurement of fasting blood glucose

FBG was determined immediately by the Accu-Chek® Performa glucometer (Roche Diagnostics, Switzerland) following the manufacturer’s instructions. All patients fasted for at least 12 hours before taking the test, and all measurements were performed by experienced trained laboratory staff.

2.6. Ethics approval

The study was approved by the Research Ethics Committee at “Al-Mustafa University College—Department of Pharmacy” (Approval number: AP011, research no.: 11, date: the 11th of January 2022). Written informed consent was obtained from all participants.

2.7. Sample size calculation

It was determined using G*Power version (3.1.9.7),^[63,64] the effect size was 0.8, α-level 0.05, β-level 0.2, with 2-tailed, and the total sample size was 52 (26 in each group).

2.8. Statistical analysis

The current study used GraphPad Prism version 10.0.1 for statistical analysis. The descriptive statistics were reported as mean ± standard deviation. The independent t test was applied to verify the significance of the difference between the studied groups. The differences between the groups were considered significant statistically when the P value was <.05 (P ≤ .05).

3. Results

The study included 52 male nonobese gasoline workers, and as illustrated in Table 1, there was no significant difference in their age, body mass index, smoking habit, education level, or monthly income.

Table 1.

Assessment of demographic parameters.

Parameters	Group A	Group B	P value
Number	26	26	—
Age (yr), mean ± SD	37.12 ± 8.47	35.85 ± 7.23	.564
BMI (kg/m2), mean ± SD	22.7 ± 1.9	22.1 ± 1.8	.262
Smoking, no. (%)	5 (19.2%)	7 (26.9%)	.510
Education level			.668
Primary or secondary	24 (92.3%)	22 (84.6%)
College	2 (7.7%)	4 (15.4%)
Monthly income			.777
Low	11 (42.3%)	10 (38.5%)
Middle	15 (57.7%)	16 (61.5%)

BMI = body mass index, SD = standard deviation.

Serum Pb was significantly higher in group A compared to group B (Fig. 1); additionally, serum levels of visfatin, MDA, insulin, FBS, and IL-1β were significantly higher in gasoline workers, while serum levels of vitamin D were significantly lower in gasoline workers, as illustrated by Table 2.

Figure 1.

Assessment of serum lead levels.

Table 2.

Assessment of investigated biomarkers and Pb.

Parameters	Group A	Group B	P value
Number	26	26	—
Pb (µg/dL), mean ± SD	14.43 ± 1.01	5.01 ± 1.41	<.001
MDA (nmol/mL), mean ± SD	6.40 ± 0.27	1.62 ± 0.21	<.001
Visfatin (ng/mL), mean ± SD	23.19 ± 0.96	3.88 ± 0.58	<.001
Insulin (mU/L), mean ± SD	22.14 ± 1.31	11.26 ± 0.75	<.001
FBS (mg/dL), mean ± SD	118.4 ± 26.1	82.7 ± 9.2	<.001
IL-1β (pg/mL), mean ± SD	330.25 ± 10.34	12.35 ± 1.43	<.001
Vitamin D (ng/mL), mean ± SD	11.99 ± 1.55	35.41 ± 3.16	<.001

FBS = fasting blood sugar, IL-1 = interleukin-1, MDA = malonaldehyde, Pb = lead, SD = standard deviation.

4. Discussion

In the present work, we demonstrated total blood Pb concentrations in gas station workers compared with healthy control and tested the relation between increased Pb concentrations and other biomedical markers. Pb is a widely recognized neurotoxic heavy metal^[65,66] found extensively in our surroundings and continues to be utilized in diverse industrial sectors.^[67] There has been a suggestion that elevated exposure to Pb may be a possible risk factor for developing VDD.^[41,42] Research conducted on children exposed to high levels of Pb has revealed a noteworthy inverse correlation between the concentration of 1,25(OH)2D and the levels of Pb in their blood.^[43,44,68]

The elevation in serum insulin and FBS in gasoline workers coupled with elevation in visfatin suggest they are at increased risk for developing DM; in addition, the elevation of serum MDA and IL-1β suggest increased inflammatory status in gasoline workers which further supports our observations of possible risk of DM in these subjects. There is a lack of studies that directly correlate Pb exposure to DM; in animal studies, some suggest a link between pre-diabetes and exposure to Pb; recent research has revealed that Pb exposure has pro-diabetic effects on the liver. These investigations specifically investigated how Pb affects hepatic metabolism in normal rats. Exposure of rats to Pb acetate in their drinking water resulted in an elevation in the enzymatic activity of hepatic gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, along with a little increase in fasting glucose levels and glucose intolerance.^[69] Mostafalou et al^[69] showed that Pb treatment ex vivo inhibited glucose-induced insulin production from islets, potentially activating glycogen synthase kinase. Tyrrell et al^[70] provided evidence that the livers of rats exposed to Pb had increased levels of transcripts for both phosphoenolpyruvate carboxykinase and glucose-6-phosphatase genes involved in gluconeogenesis. They also proved that this effect could be reproduced in vitro using hepatoma cell lines treated with Pb. In another animal study, exposure to low levels of Pb can raise glucose production in the liver by impacting important enzymes involved in gluconeogenesis; this can ultimately Pb to higher glucose levels in the blood after fasting and the development of hyperglycemia.^[71] These studies suggest a possible association between Pb exposure and increased risk pre-diabetic and DM. Our findings were in line with these previous studies, and we suggest it involved increased inflammatory stress in humans exposed to Pb.

A notable decrease in serum 1,25(OH)2D concentration was seen in children with blood Pb levels ranging from 33 to 55 μg/dL. The decline in serum 1,25(OH)2D was particularly notable among children with blood Pb levels exceeding 62 μg/dL, indicating a relationship between Pb exposure and vitamin D levels that depends on Pb dosage.^[43] The consumption of Pb in animal experiments decreased the serum concentration of 1,25(OH)2D and inhibited vitamin D-dependent intestinal calcium transport in rats.^[72] There is evidence to show that elevated blood Pb levels may lead to interference with the renal hydroxylation process of 25(OH)D by 1-α-hydroxylase, resulting in the synthesis of the biologically active form of vitamin D, known as 1,25(OH)2D.^[45] As a result, the decrease in the synthesis of serum 1,25(OH)2D caused by Pb toxicity is accompanied by a decrease in calcium levels and a rise in serum parathyroid hormone levels. Therefore, Pb disrupts multiple vitamin D actions essential for maintaining calcium homeostasis and regulating metabolism in diverse tissues and organs.^[46,47,73]

Lead poisoning is attributed to 3 primary mechanisms: blockage of the heme synthesis pathway, ionic mimicry,^[74,75] and oxidative stress.^[14,76] The process by which Pb induces hypertension, infertility, and liver and kidney damage is well-established and involves oxidative stress.^[14] Oxidative stress arises from an inequilibrium between the levels of free radicals throughout the body (or individual cells) and the body’s capacity to neutralize the reactive intermediates produced by these free radicals. Hence, an elevation in the levels of free radicals or a reduction in the presence of antioxidants might lead to oxidative stress. The presence of increased levels of ROS or a reduction in the concentration of antioxidant sulfhydryl-rich compounds, such as GSH, can serve as indicators of oxidative stress.^[77]

Several mechanisms exist by which exposure to Pb might alter the equilibrium between GSH and GSSG, ultimately leading to oxidative stress. Pb forms complexes with the sulfhydryl groups in proteins, resulting in reduced GSH reserve levels.^[78–80] Additionally, Pb exhibits potent inhibition of d-aminolevulinic acid dehydratase, perhaps resulting in elevated levels of its substrate, δ-aminolevulinic acid. The δ-aminolevulinic acid undergoes auto-oxidation, producing ROS^[81]; the detoxification process involves the conversion of reduced GSH to GSSG.^[82] A positive correlation exists between occupational exposure to Pb and elevated MDA levels, an oxidative stress biomarker resulting from lipid peroxidation. Additionally, there is evidence of reduced amounts of GSH or potential disruptions in its synthesis processes in individuals exposed to Pb.^[83–85]

4.1. Study limitations

The observational nature of the study will limit the generalization; in addition, nationwide surveillance is needed regarding Pb exposure to the general population. To correlate Pb exposure to its serum level, the measurement of its concentration in the environment was not performed, which would give a clearer picture.

5. Conclusion

This study presents evidence supporting a positive association between blood Pb levels and increased inflammatory and oxidative stress markers. Lead exposure increases serum insulin and fasting blood sugar, which could be linked to the direct toxic effect of Pb exposure, which causes inflammation and oxidative stress damage to the pancreas.

Author contributions

Conceptualization: Ahmad Tarik Numan, Nada Kadum Jawad, Hayder Adnan Fawzi.

Investigation: Ahmad Tarik Numan, Nada Kadum Jawad.

Methodology: Ahmad Tarik Numan, Nada Kadum Jawad, Hayder Adnan Fawzi.

Project administration: Ahmad Tarik Numan, Nada Kadum Jawad.

Resources: Ahmad Tarik Numan, Nada Kadum Jawad.

Writing—original draft: Ahmad Tarik Numan, Nada Kadum Jawad, Hayder Adnan Fawzi.

Writing—review & editing: Ahmad Tarik Numan, Nada Kadum Jawad, Hayder Adnan Fawzi.

Data curation: Nada Kadum Jawad, Hayder Adnan Fawzi.

Software: Nada Kadum Jawad, Hayder Adnan Fawzi.

Formal analysis: Hayder Adnan Fawzi.

Supervision: Hayder Adnan Fawzi.

Validation: Hayder Adnan Fawzi.

Visualization: Hayder Adnan Fawzi.