Oxidative stress and adverse cardiovascular effects among professional divers in Egypt

Abstract Professional divers are exposed to unique multifactorial hazards in their working environment and adverse cardiovascular effects such as ischemia, arrhythmia, stroke, and death are associated with professional diving. Cardiovascular events are aggravated by diving-induced oxidative stress and account for one-fourth of diving fatalities. This study aimed to measure oxidative and cardiovascular stress in a group of professional divers in Alexandria, Egypt using a panel of biomarkers. A comparative cross-sectional study was conducted between June 2017 and May 2018 at the General Naval Hospital in Alexandria. A total of 50 professional divers and a comparison group of 50 marine seafarers sharing similar maritime environments were enrolled in the study. Participants were clinically evaluated by electrocardiography (ECG) and plasma measurement of trace metals (Fe+, Cu+, and Zn+), electrolytes (Na+, K+, Ca+), and oxidative stress biomarkers (OSBMs; MDA, TAS, GST, GSH, GR, GPx, SOD, and CAT). Significant ECG abnormalities including short corrected QT interval, sinus bradycardia, left ventricular hypertrophy, early repolarization, first-degree heart block, and intraventricular conduction defect were identified among divers. Biochemical analyses revealed high mean levels of FBG [89.0 ± 12.46 vs. 100.5 ± 29.03 mg/dl], LDH-C [41.46 ± 4.01 vs. 39.34 ± 4.34 mg/dl], electrolyte imbalance [higher Na+ (9.44 ± 0.52 vs. 9.19 ± 0.60 mmol/L), and lower Ca+ (141.72 ± 3.53 and 143.26 ± 3.99 mmol/L)], disturbed trace metals [Fe+ and Zn+ (101.1 ± 38.17 vs. 147.6 ± 38.08 and 85.52 ± 27.37 vs. 116.6 ± 21.95 µm/dl, respectively), higher Cu+ (271.3 ± 75.01 vs. 100.8 ± 30.20 µm/dl)], and higher OSBMs (high MDA and reduced CAT, GPx, GSH, GR, and GST enzyme levels) among professional divers compared to the marine seafarers (t-test p < 0.05). Oxidative stress and trace metal imbalance are associated with the pathophysiology of cardiovascular disease; this association, together with electrophysiological changes of ECG may serve as biomarkers for cardiovascular risk assessment in diver periodic medical examinations.


Introduction
Divers are employed in a wide range of activities, and share unique hazards within the working environment (Mitchell and Bennett 2008). Diving exposes a body to an unnatural hyperbaric environment which can lead to increased gas dissolved in the tissues (Pendergast and Lundgren 2009). In addition to gas toxicity associated with the diving environment divers are exposed to various stress factors including excess physical demands, hemodynamic changes, and thermal and mental stresses. Disorders affecting divers are not limited to gas toxicity but can affect locomotion, respiration, circulation, and the kidney excretory and nervous systems (Levett and Millar 2008). Cardiovascular disease (CVD) has been identified as a possible contributing event to diving-related fatalities (Denoble et al. 2008;Bove 2011;Mitchell and Bove 2011;Edmonds and Caruso 2014). Oxidative stress (OS) and chronic elevation of reactive oxygen species (ROS) is an additional contributing risk for cardiovascular disorders among divers (Tirapelli 2020). Increases in free radicals of nitrogen and oxygen species damage lipoproteins, DNA, and lipids and proteins of cell structures. Thus, OS is mainly assessed by measuring oxidative stress biomarkers (OSBMs) which include malondialdehyde (MDA), glutathione (GH), reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GRH), catalase enzyme, and superoxide dismutase (SOD) (Ceconi et al. 2003).
Likewise, trace metals are key to the pathogenesis and evolution of in CVDs. The presence of Iron (Fe þ ), Copper (Cu þ ), and zinc (Zn þ ) in the blood is crucial for cardiovascular health and fitness. Trace metal levels in the blood are disturbed in established CVDs (Valko et al. 2005;Shokrzadeh et al. 2009) and are emerging as novel markers for cardiovascular risk assessment of divers due to trace metal correlation with CVDs and OS (Valko et al. 2005(Valko et al. , 2007.
The classical diver fitness and risk assessment in the pre-placement and periodic exam utilize conventional tools stressing lipid risk factors, and on some occasions, cardiac stress factors without considering early biomarkers that may indicate an increased risk of developing cardiovascular diseases among professional divers.
Accordingly, the investigators involved in this study proceeded to analyze the diving effect on some OSBMs and trace elements that may affect fitness and cardiovascular risk among professional divers compared to marine seafarers.

Study setting, design, and population
A comparative cross-sectional study was conducted between June 2017 and May 2018 at General Naval Hospital in Alexandria at clinics authorized to conduct diving medical examinations. The target population comprised professional male divers, fishermen divers, and freelance divers at different governmental and private organizations in Egypt. Accordingly, the investigators recruited a representative sample of eligible professional divers and a second group of nondiver marine workers matched in socio-demographics and sharing the maritime work environment with no exposures to workplace diving hazards. Professional divers and marine seafarers seek medical fitness examinations at the General Naval Hospital in Alexandria as a prerequisite for obtaining and/or renewal of their marine license. Eligibility criteria comprised male divers from 30-45 years of age, certified as professional divers with a diving license of fewer than 5 years, had accomplished at least two dives/month within decompression limits during the last year before examination, and having a complete signed logbook recording: (i) the diver medical fitness certificate (valid and signed by an authorized diving medical physician), and (ii) diving history that included the number of dives, maximum depth, maximum duration, diving bottom duration, type of diving gas used, and diving plan. Divers and mariners with new overt CV symptoms, diabetes mellitus (DM), or any chronic inflammatory diseases such as rheumatoid arthritis, osteoarthrosis, or any other musculoskeletal disorders were excluded from the study.

Sampling
Using G Ã Power software (V. 3.0.10, Heinrich Heine Universit€ at Dusseldorf, Germany), and based on a previous study of CVR factor assessment in professional divers (Pougnet et al. 2012), the minimum required sample size was 46 for each group. Fifty participants were ultimately enrolled in each group. All eligible professional divers and seafarers fulfilling the inclusion criteria and accepted to participate in the study were consecutively enrolled until the desired sample size was achieved.

Clinical and laboratory assessments
All enrolled participants were interviewed to record occupational and medical history using a predesigned questionnaire to collect background information on sociodemographics, lifestyle, type and duration of professional activity, and family and past medical history for relevant medical conditions to identify comorbidities.
The smoking index was calculated for current smokers by multiplying the number of cigarettes per day and the number of smoking years (Centers for Disease Control Prevention [CDC] 2016). Subjects were classified into either smokers (those who had smoked more than 100 cigarettes in their lifetime and currently still smoking) or nonsmoker (those who had not smoked more than 100 cigarettes and currently not smoking).
All divers were clinically evaluated with complete physical examinations (anthropometric measurements, complete general, and systemic examination). Systemic clinical examination was done with a special emphasis on the cardiovascular system, which included resting heart rate, respiratory rate, and blood pressure measurements according to the standard procedures (Colantonio et al. 2018).

Electrocardiography
A 12-lead surface electrocardiogram (ECG) was completed for all patients in the supine position using an ECG device. The 12 lead ECG was recorded at a paper speed of 25 mm/sec with 1 mV/cm standardization. The following data were noted: Rate/min, P-R interval (msec), QRS complex (msec), QT wave (msec), R-R minimum, and R-R maximum which are measurements of the long lead II during deep inspiration and the Sokolov of the precordial chest leads, respectively.
The corrected QT wave interval was calculated according to Equation (1), which was developed by Chenoweth et al. (Bazett 1920;Israel et al. 2005;Chenoweth et al. 2018): where QT ¼ time from the beginning of the QRS complex (representing ventricular depolarization) to the end of the T wave, resulting from ventricular repolarization; and ͱ(R-R Interval) ¼ square root of the time elapsed between two successive R-waves of the QRS signal on the ECG. The main ECG criteria for left ventricular hypertrophy (LVH), including Sokolov-Lyon and Cornell Voltage indices, were calculated for all included individuals. The LVH was calculated using Equation (2) (Tamama et al. 1998;Pougnet et al. 2012;Tocci et al. 2017): (2) where S 1/2 ¼ amplitudes of the S-wave in V1 or V2 (whichever is greater) and R 5/6 ¼ amplitude of the Rwave in V5 or V6 (whichever is greater).

Blood sampling
Five milliliters of venous blood was collected aseptically from each participant in a vacutainer tube containing citrate or EDTA as an anticoagulant. Tubes containing blood were centrifuged at 700-1,000 Â g for 10 min at 4 C. The top yellow plasma layer was pipetted off without disturbing the white buffy coat. The white buffy layer was then removed and discarded.
The RBCs were lysed in four times the volume of ice-cold HPLC-grade water. The lysate was centrifuged at 100,000 Â g for 15 min at 4 C. The collected plasma and erythrocyte lysate samples were stored on ice until assaying was completed on the same day or the sample was frozen at À80 C to preserve samples for analysis within 1 month.

Determination of trace metal blood concentrations
Plasma trace metal levels of Fe þ , Zn þ , and Cu þ were analyzed by the colorimetric technique described by Kim et al. (2013).

Statistical data analysis
Data were analyzed using the IBM SPSS software package (Version 20.0, IBM Corp., Armonk, NY). The Kolmogorov-Smirnov test was used to verify the normality of the distribution of the different variables. Qualitative data were described using number and percent. Quantitative data were described using mean and standard deviation (SD). The chi-square test was used for categorical variables to compare different groups. Fisher's Exact test (FET) was used as a correction for chi-square when more than 20% of the cells have an expected count of less than five. For normally distributed quantitative variables, the t-test was used to compare the two studied groups. Pearson coefficient was used to correlate between two normally distributed quantitative variables. The Mann Whitney U test was used for abnormally distributed quantitative variables to compare between two studied groups. Wilcoxon signed ranks test was used for abnormally distributed quantitative variables to compare between two time periods. The significance of the obtained results was judged at the 5% level.

Sociodemographics and clinical data of the enrolled divers and seafarers
The mean age of divers was 36.5 ± 6.1 years, while that of the marine seafarers was 37.0 ± 5.1 years (p > 0.05). The average employment duration for divers was comparable to that of the marine seafarers (13.1 ± 8.4 vs. 13.0 ± 7.6 years, respectively).
Family history of CVD and/or DM was more frequently reported in the diver group than the seafarers although the differences between the two groups for both conditions were not statistically significant (p > 0.05).
A considerable number of the professional divers and marine seafarers were smokers, although the pattern of smoking did not differ significantly between the two studied groups (p > 0.05).
The mean (±SD) BMI of the professional divers was 26.7 ± 3.7 with the majority being overweight. Type I obesity was prevailing among marine seafarers as evidenced by higher BMI, waist circumference, and waistnheight ratio compared to the professional divers (p < 0.05). On the other hand, the divers showed a statistically significant higher mean SBP compared to the marine seafarers [131.2 ± 14.06 vs. 124.3 ± 15.84, respectively (p < 0.05)]. See Table 1.

Biochemical changes among study participants
The mean FBG level differed significantly amongst divers and seafarers [89.00 ± 12.46 vs. 100.5 ± 29.03 mg/dl (p > 0.05)], though the levels were still within normal reference ranges (56-110 mg/dl). Likewise, renal function tests (blood urea, creatinine, and uric acid levels) and liver function tests (SGOT, SGPT, and serum bilirubin) were comparable between the two groups and within normal reference ranges (p > 0.05).
In the lipid profile, the mean plasma levels of TC, TG, and LDL-C were comparable between the professional divers and seafarers (p > 0.05). On the other hand, the professional divers showed higher mean HDL-C levels compared to the seafarers [41.46 ± 4.01 vs. 39.34 ± 4.34, respectively (p < 0.05)]. See Table 2.

Biomarkers of oxidative stress
Regarding lipid peroxidation, the mean MDA level was high in both groups compared to its reference range (0.67-2.44 mmL), although it was significantly higher among the professional divers compared to the seafarers [13.54 ± 4.08 vs. 6.31 ± 2.66 mmL (p < 0.001)].
As for the antioxidant system, the TAS was not increased in both groups. The mean TAS level remained within the normal reference range (0.5-2.0 mmL), although it was significantly lower in the divers than in the marine seafarers [0.84 ± 0.43 vs. 1.40 ± 0.45 mmL, respectively (p < 0.001)]. The mean CAT, GPx, GSH, GR, and GST enzyme levels were significantly reduced in the professional divers (p < 0.001). On the other hand, the mean SOD level was high in both groups compared to the reference range (100 U/mL), although it was significantly higher among marine seafarers compared to the professional divers [140.5 ± 27.59 vs. 243.2 ± 40.19 U/mL (p < 0.001)]. See Table 2.

Trace metals
The mean Fe þ level was significantly lower among professional divers than in marine seafarers

Discussion
In this study, the investigators used a multi-marker approach to assess cardiovascular disease and OS in professional divers. The panel of biomarkers included some OSBMs, trace metals, electrolytes, and electrophysiologic changes in the ECG.
The study population of professional divers and seafarers were matched with regard to age, duration of employment, history of smoking, and family history of CVDs and DM. Seafarers were commonly  overweight or obese, although the percentages were also high amongst divers. This was also reported in a study completed in France on 200 professional divers where 43.6% of divers were overweight or obese (Pougnet et al. 2012). In a study measuring BMI in 1,115 German and Italian sailors, approximately 40% of the evaluated subjects were overweight, and more than 10% of them were obese (Nittari et al. 2019). The relative differences in BMI could reflect the increased physical activities inherent in professional diving and compared to those of seafarers.
The BP was elevated in most participants albeit with a higher percentage among divers, which is similar to findings from other studies where the percentages of high BP among divers varied between 6.5% and 64.0% (Pougnet et al. 2012;Åsmul et al. 2017). Increased prevalence of high SBP was also reported among seafarers in Denmark (44.7%) (Tu and Jepsen 2016) and Germany (33.8%) (Oldenburg et al. 2008). Increased BP among divers may be due to the higher OS associated with diving.
Apart from HDL-C, lipid profile measurements did not differ significantly between the two study groups where levels remained within normal ranges, contradicting reports in other studies that increased TC in 50% of professional divers (Pougnet et al. 2012). Another study reported higher TG levels among 41.6% of seafarers (Oldenburg 2014).
Liver and kidney function tests together with uric acid were within normal ranges among the two groups indicating that there were no kidney or liver conditions that could influence the other assessed laboratory parameters, particularly OSBMs and trace metals which can indicate potential liver and kidney disease (Halliwell and Cross 1994;Romero et al. 1998). Likewise, normal mean FBG was significantly lower among divers than seafarers and may be a reflection of the excess physical demands of divers, and to a lesser extent, of the diving environment as postulated in similar studies (Dear et al. 2004;Sponsiello et al. 2018).
The ECG is necessary for early diagnosis of cardiovascular pathology. Few studies addressed qualitative Table 3. Quantitative and qualitative electrocardiogram changes among the enrolled divers and marine seafarers. changes in the ECG. In the present study, professional divers showed five times the odds of having electrophysiological changes compared to marine seafarers. Sinus bradycardia, LVH, early repolarization, firstdegree heart block, and intraventricular conduction defect were obvious changes. High ambient pressure and increased gas densities associated with diving can lead to cardiac decompensation. Sinus arrhythmia is frequent in athletes as a result of strenuous exercise whereas other changes in ST segments and T-wave are considered to be pathological (Kiss et al. 2015). In a study of 225 randomly selected professional divers, significant ECG abnormalities such as incomplete IRBBB, right QRS axis deviation, sinus tachycardia, early repolarization, ventricular extrasystoles, ST elevation, sinus arrhythmia, and T-wave inversion were reported (Gunes and Cimsit 2017). Electrophysiological changes are important clues and are more likely to precipitate or aggravate arrhythmias in divers and hence more relevant etiological factors for cardiac-related deaths rather than ischemic coronary vascular changes (Greenland et al. 2010;Denoble 2013;Shenasa and Shenasa 2017;Bo€ asson et al. 2019). Including electrophysiological changes in CVR assessments of divers will help also in risk stratification when an assigned score can reflect the risk level associated with the procedure. No other studies described corrected QT waves except a single study by ZiaZiabari et al. (2019). ZiaZiabari et al. (2019) reported significant changes in pulse rate, corrected QT, and T-wave before and after diving but did not define a cutoff point suggestive of the repolarization instability. ECG changes were also found to be a more specific biomarker as OS changes could occur in other physiological and pathological conditions (Tocci et al. 2017). The use of ECG changes as a biomarker may be especially useful due to widespread ECG availability, simple interpretation, and cost-effectiveness, and should be a preferred biomarker in CVD risk assessment (Tocci et al. 2017).
Evidence for increased OS was found in both studied groups as manifested by abnormal levels of OSBMs. This was more obvious among divers compared to seafarers and came agreed with previous studies that identified an association between increased OS and increased cardiovascular risks and hypertension (Birben et al. 2012;Ho et al. 2013;Frijhoff et al. 2015;Bastani et al. 2018). Other studies showed that the assessed CVR was higher than the OS levels (Poredo s and Je zovnik 2015). Meanwhile, the current results support the postulation that diving and physical exertion, including exercise, increase OS in divers (Gorman et al. 2009; Perovi c et al. 2014).
Remarkably, MDA, SOD, GST, and TAS were more sensitive indicators of OS than other biomarkers when demonstrating the highest percentage of changes compared to normal reference values.
The role of trace metals and OS in the pathogenies of CVDs is well established (Momtaz et al. 2000;Osredkar and Sustar 2011;Nagarajrao 2014). Also, OS is linked to increased levels of Cu þ and decreased levels of Fe þ and Zn þ (Sarkar et al. 2007;Osredkar and Sustar 2011;Marreiro et al. 2017). This was in concordance with the present work, in which levels of Fe þ and Zn þ were significantly lower in divers compared to seafarers, and levels of Cu þ were significantly higher. Few researchers have mentioned the effect of diving on blood concentrations of trace metals, especially the saturation inherent in deep diving which affects the hemoglobin iron level (Nakabayashi et al. 1991).
In this study, mean serum electrolyte (Ca þ , Na þ , and K þ ) levels among the two groups were within normal reference values, although higher levels of Ca þ and lower levels of Na þ were observed in the professional divers compared to the seafarers. A recent study showed that high blood Ca þ levels may increase arterial wall stiffness and subsequently the 10-year CVD risk as assessed by the Framingham score (Park and Lee 2019), which might represent another factor that can lead to increase in the CVD risk. Indeed, hyponatremia may be attributed to repeated physical activity associated with diving and the physical effects of diving, which agree with the findings of Luhker et al. (2017) and Rosner and Kirven (2007) that identified exercise-associated hyponatremia and disturbed acid-base homeostasis after sustained physical exertion during athletic events.

Conclusion
In conclusion, levels of OSBMs as well as trace metal imbalance were found above previously identified benchmarks for CVD risk in divers. The electrophysiological changes of cardiac activities detected by ECG are powerful CVD biomarkers. Furthermore, OS and trace metal levels are linked to the pathophysiology of CVDs and together with electrophysiological changes of ECG may be appropriate for use as novel biomarkers for CVR assessment.

Limitations of the study
Although OS and trace metals are useful biomarkers in the present context, they are nonspecific and can be confounded by different exposures as well as dietary, physical, and lifestyle patterns. Analysis of variables related to the occupational activity such as several dives, depth, duration, and type of gas was out of the scope of this manuscript and will be presented separately. A multivariate analysis considering these covariates would give a clearer picture of the relationship between OS and CVD risk in divers, although this was not possible to run in the current study given the relatively smaller sample size to the number of variables included.