Oxidative stress and motion sickness in one crew during competitive offshore sailing

Competitive Offshore Ocean Sailing is a highly demanding activity in which subjects are exposed to psychophysical stressors for a long time. To better define the physiological adaptations, we investigated the stress response of subjects exposed to 3-days long ocean navigation with disruption of circadian rhythms. 6 male subjects were involved in the study and provided urine and saliva samples before setting sail, during a single day of inshore sailing, during 3-days long ocean navigation, and at the arrival, to measure oxidative stress, cortisol, nitric oxide metabolites (NOx) and metabolic response. Motion Sickness questionnaires were also administered during the navigation. The crew suffered a mean weight loss of 1.58 kg. After the long navigation, a significant increase in ROS production and decrease in total antioxidant capacity and uric acid levels were observed. Lipid peroxidation, NO metabolites, ketones, creatinine, and neopterin levels were also increased. Furthermore, a significant increase in cortisol levels was measured. Finally, we found a correlation between motion sickness questionnaires with the increase of NOx, and no correlation with cortisol levels. Physical and psychological stress response derived from offshore sailing resulted in increased oxidative stress, nitric oxide metabolites, and cortisol levels, unbalanced redox status, transient renal function impairment, and ketosis. A direct correlation between motion sickness symptoms evaluated through questionnaires and NOx levels was also found.


Methods
Experimental design. This observational study was carried on in November 2020 during an OOS racingoriented training that included a theoretical part ashore, a full training day of inshore sailing, and 3 days of nonstop OOS roughly between the latitudes of Gibraltar and Lisbon. The crew sailed in a Class 40 (ITA 84) racing yacht and, during the navigation, was divided into two groups alternating rest and duty shifts every 3 h. Figure 1 depicts the study protocol and samplings. Urine and saliva samples and anthropometric measurements were obtained ashore during the theoretical part (PRE) and approximately two hours after the end of the navigation (POST) to be as accurate as possible. Further, two urine samples were obtained during the single day of inshore navigation (Training) and three times a day during OOS (Sailing).
Subjects. This study involved six male sailors: the skipper (S.F.)-a professional sailor with experience in ocean solo races-and five recreational sailors with good expertise in seamanship The total number of subjects was determined by the maximum crew that the boat could support and by chance all the participants to this training were healthy male. Their characteristics are reported in Table 1.
Navigation. The offshore navigation lasted 3 days, during which the crew sailed into the ocean for a total of 420 miles, with a top speed of 14.89 kn.
During the first day and night, the atmospheric conditions were challenging. Swell of 3/3.5 m Significant Wave Height (SWH) from NW and wind from 5 to 15 kn from S-SE resulted in a boat's inconvenient motion. During the day, the wind increased between 25 and 40 kn in gusts as sailors encountered two significant squalls and had to flee downwind. During the night, sailors were forced to maintain a 70° true wind angle (TWA) sailing upwind to cope with waves, and the wind speed increased up to 45 kn. After the first day, the crew was objectively stressed. These harsh conditions induced major seasickness and vomiting in one crew member, with a total inability to work on deck. This subject started to recover only at the end of the navigation, during which he never ate and vomited many times without being able to drink and rehydrate. Two other people vomited but were not impaired at work. Liquid reintegration started the day after. On the second day, the conditions changed, with waves height reduced to 1-2 m SWH. The boat headed downwind, hoisting a code zero sail, maintaining the boat flat at an average speed of 9-10 kn with 15-25 kn of wind speed. The navigation remained stable until the end of the navigation in Lisbon on the third day.
Motion Sickness Questionnaire. To study motion sickness, previously validated Global Sickness Rating Scale (GSRS) 27 and Motion Sickness Questionnaire (MSQ) 14 were used. They have been administered once per day during the Offshore Sailing, in the evening, at 9 PM, according to the 3 h shifts. www.nature.com/scientificreports/ Saliva and urine collection. Approximately 1 mL of saliva was obtained before and after the training and collected in Salivette devices (Sarstedt, Nümbrecht, Germany) at 8 AM. The subjects were trained on the correct use as previously reported 28,29 . Urine samples were collected by voluntary voiding in a sterile container before and after the training and every day during the training and navigation at 9 AM, 3 PM, and 9 PM according to the 3 h shifts. All samples were stored at 4 °C in a portable cooler on board and during the transport back to the laboratory. The specimens were then stored in multiple aliquots at − 20 °C until assayed and thawed only once before analysis.

ROS and TAC .
An X-band electron paramagnetic resonance spectroscopy (9.3 GHz) (E-Scan, Bruker Co., MA, USA) was used to detect ROS production and TAC values. Saliva samples were stabilized at 37 °C using a Temperature and Gas Controller "Bio III" unit (Noxigen Science Transfer & Diagnostics GmbH, Germany), interfaced with the E-Scan. ROS production and TAC assessment methods were previously described 28,30 . Samples were analyzed in triplicate.
Cortisol. The concentration of free cortisol in the saliva was quantitatively determined through ELISA method according to the manufacturer's protocol (COR(Cortisol) ELISA Kit; FineTest, Wuhan Fine Biotech Co.) as previously described 31 . www.nature.com/scientificreports/ 8-Isoprostane. Lipid peroxidation was assessed in urine by competitive immunoassay measuring 8-isoprostane concentration (8-iso-PGF2α) (Cayman Chemical, USA). The method was previously described 32 .

PRE
NO metabolites. NO metabolites (NOx = NO 2 + NO 3 ) levels were assessed in urine by a method based on the Griess reaction 33 , using a commercial kit (Cayman, BertinPharma, Montigny le Bretonneux, France) 28,33 .
Every assessment was carried out in duplicate and read by a microplate reader spectrophotometer (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland).
Urine standard analysis. The Urine Test Strips (Combi screen 11sys PLUS, GIMA, Gessate, Milan, Italy) were used to semi-quantitative determinations of bilirubin, urobilinogen, ketones, proteins, blood, pH, leukocytes, and specific gravity/density in urine. The tests were performed in duplicate.
Statistical analysis. Statistical analysis was performed using the GraphPad Prism package (GraphPad Prism 9.0.1, GraphPad Software Inc., San Diego, CA) and SPSS statistics software (IBM corporation). Data are presented as mean ± SD. Statistical analyses were performed using: non-parametric tests, Wilcoxon matchedpairs signed-rank test for independent samples (ROS and TAC and cortisol in saliva), due to the small sample size for compared pre vs. post and ANOVA repeated measures, with Dunn's multiple comparison tests to further check the among-groups significance. p < 0.05 was considered statistically significant..dCohen was used for calculating the size effect, and Confidence Interval 95% for d Cohen was calculated.
Ethics approval. This research study was approved by the Ethical Committee of University of Milan, Italy (Aut. n° 37/17). All procedures conformed to the standards set by the 1964 Declaration of Helsinki and its later amendments.

Consent to participate. Informed consent was obtained from all individual participants included in the study.
Consent for publication. All authors have read the manuscript and expressed their consent for the publication.

Results
A significant difference (p < 0.05) was observed in weight (kg) between Pre and Post ( Table 1). All crew members suffered a loss of weight (Mean weight loss: 1.58 kg).
No significant differences were recorded in GSRS for different items and MSQ during the 3 days of offshore navigation (see Tables 2, 3).

Discussion
To our knowledge, this is the first study to investigate oxidative stress on urine and saliva sampled from nonprofessional sailors during OOS and possible correlation with motion sickness. This setting is particularly challenging, with rapid changes in terms of environmental conditions and circadian rhythms. According to the results, subjects exposed to OOS suffer a significant multifactorial increase in oxidative stress and cortisol. A small number of studies considered modifications in cortisol levels in sailors and seafarers. Oldenburg et al. found that cortisol awakening levels were highly dependent on subjective stress perception and work type. Mental work was also associated with higher cortisol levels than physical work 35 . This is confirmed by cortisol levels found in maritime pilots, increasing their tasks' difficulty 36 . Some studies' results reflect that seafarers' cortisol levels are higher in port stays than at sea, probably because of the break of a working routine found during days at sea 35,37 . Liberzon et al. found that cortisol response at awakening in the crew increased with navigation time and was not correlated with sleep duration or patterns 37 . Other confined environments in which the crew suffers a sudden and prolonged change in circadian rhythms and psychosocial stress are, for example, military ships, submarines, or spaceships. In these environments, a flattening of the standard cortisol fluctuation profile has also been recorded under stressful conditions 38,39 . Similar results were obtained by Gunnarsson et al. on ocean sailors studied for a more extended period during an offshore regatta. They also reported an initial increase in cortisol levels at the beginning of the navigation, with a flattening of the fluctuation when sailors reached the regularization of the shift regimen 12 . Our study found a significant increase in cortisol levels in sailors after 3-day-long offshore navigation compared to their basal level (+ 15%, see Fig. 2H). These results may be related to the significant stressors that maritime personnel and offshore ocean sailors have to endure, particularly fatigue and poor sleep quality. From a physiological point of view, sleep disturbances induce a decrease in physical and cognitive performance in sailors 8 and a disruption of normal cortisol secretion, causing the activation of pro-inflammatory pathways 40,41 . Physical exercise can also induce a modification in cortisol secretion 42 . During offshore sailing, a basal level of muscle activation is needed to cope with instability and to aid  Table 3. Motion Sickness Questionnaire (MSQ). Mean (± SD) values of the investigated variables. Urine standard parameters are reported in Table 4. A significant increase in urinary ketones levels was detected during the navigation. pH and bilirubin values also increased but did not reach statistical significance. www.nature.com/scientificreports/ thermoregulation, but short bursts of anaerobic exercise are required in all the maneuvers 1,[17][18][19] . It is, therefore, reasonable to think that these aspects also contribute to cortisol levels alterations. During day one, three subjects suffered from motion sickness and had vomiting episodes. Although cortisol is known to correlate with acute nausea and vomiting 43,44 , we found no significant correlation between this hormone and the seasickness scale questionnaires administered 14,27 . Nonetheless, we have been able to measure nitric oxide metabolites (NOx) levels throughout the navigation. Nitric oxide is involved in many gastrointestinal mucosal mechanisms 26 , and previous studies found a correlation between salivary and serum NO levels with vomiting syndromes and Gastroesophageal Reflux Disease (GERD) [43][44][45][46] . Nausea caused by motion sickness is also characterized by gastric dysrhythmias 47 . In accordance with these studies, we found a significant linear relationship between NOx levels and GSRS during the first day of navigation, during which the subjects suffered the most intense motion sickness (Fig. 3). Table 4. Urine standard analysis. Mean (± SD) values of the investigated variables in the urine test strip in the sailors. Changes in urine standard urinalysis referred to PRE are shown. Statistically significant difference at p < 0.05 (* symbol).  www.nature.com/scientificreports/ During inshore regattas, short bursts of high-intensity activity are described 18,19,48 . However, even if data regarding physical effort during offshore sailing seem comparable with inshore activity 17 , the evidence is scarce, difficult to obtain, and limited to measuring the effects of energy expenditure and physical effort activity after the race. Weight loss, fat percentage decrease, lower limb strength, and muscle mass reduction are often reported 1,3,4,6,49 . ROS production is enhanced by exercise [21][22][23]29,50 . In particular, anaerobic exercise can induce prolonged oxidative stress up to 24 h after the effort 51,52 , which is then balanced by an enhanced antioxidant response 21,23,53 . In America's Cup sailors, oxidative stress markers after the race were higher than their baseline levels, especially in crew members involved in high-intensity physical work 54 . Our study is the first to analyze oxidative stress markers during OOS. Our results show a significant increase in ROS production after the navigation. The imbalance between the ROS production rate (about + 100%) and the antioxidant scavenging (− 12%, see Fig. 2A-C) reflected the increase in the oxidative stress-related damage to lipids (+ 87%; see Fig. 2D). Oxidative stress is highly involved with inflammation and endothelial dysfunction in developing chronic cardiovascular diseases 55,56 . Even though more evidence should be produced on ocean sailors, the effects of oxidative stress exposition on seafarers can be a potential cause of their higher cardiovascular risk and mortality rate for coronary heart disease [57][58][59] .
Neopterin and creatinine concentration can increase during systemic oxidative stress, as shown in some studies 23,28,29,60 . Even if a decrease in kidney function can be a hint of organ damage during endurance sports, it is often the result of many physiological responses to stress and physical demands 21,22,61 . In our study, an increase of evaluated biomarkers concentrations was observed during and post-offshore sailing and was associated with ROS production. In any case, this study did not assess the chronic or long-term effects of offshore sailing. Mainly referred to kidney activity, the subjects manifested a temporary "impairment of renal function" as a likely physiological or adaptive response to dehydration. This could also be linked to significant weight loss (see Table 1) and vomiting, which changes ketones concentration, pH, and specific density (see Table 4). Ketones increase could also hint at how athletes' metabolism copes with high energy demand and stress. Their production is stimulated by low insulin, high glucagon, and epinephrine concentrations, suggesting a shift to metabolic efficiency and fuel sparing of the organism exposed to endurance exercise and fasting 62,63 . They are also second messengers for many pathways, such as food intake stimuli 64 . The ketogenic regimen is also related to the increase of lipid metabolism 65 which in the case of OOS is often associated with the decrease of body fat percentage and weight loss 4,6,49 . Considering that sailors are exposed to harsh environmental conditions and that motion sickness and working rates can influence nutrition habits during a race 4 , it is of utmost importance to maintain an adequate water intake during navigation to prevent renal damage and to keep proper caloric intake to sustain physical performance.

Limitations and conclusions
As for other studies 4,6,12,17 that focus on OOS, we found many difficulties in producing reliable data and scientific evidence. The researcher himself, which was part of the crew, had to take part in the strenuous activity schedule, the space for medical devices and samples on board is limited, invasive procedures are complicated to perform because of continuous motion, electronic devices cannot be charged because electrical power is limited and needed for navigation. The complexity of this environment often results in a lack of reliable literature 1 . Therefore, we chose to obtain urine and saliva samples because of the limited logistic disadvantages of these samples.
A limitation of this study is the lack of data on the quality and duration of sleep. This might have influenced the cortisol level, but Liberzon et al. found no connection between sleep and cortisol levels 37 , and other OOS experiments show data similar to ours 12 .
Some previous experiences in literature 66 found a correlation between cortisol levels and motion sickness. We speculate that probably due to the low number of investigated subjects and saliva samples, and because we didn't measure cortisol levels immediately after vomiting episodes, we found no significant correlation between motion sickness questionnaire results and cortisol levels. Therefore, to be more confident on these results our methods could be implemented. Another factor to be considered could be that we investigated only male subjects, and as previously reported by Meissner et al., the cortisol level changes in saliva in male patients could not be significant. Cortisol response in motion sickness, as they suggest, should be corrected for the hour of the day, gender, and basal cortisol levels 67 . Moreover, high variability was observed in oxidative stress markers, cortisol levels, and motion sickness scales between the same sailors on various days.
Another limitation is that we have not been able to obtain information on sailors' cardiovascular and metabolic activity during the navigation, even though they have been described in other similar and comparable studies 17,18 . In the future, we hope we will be able to implement our methods and obtain this data in a similar environment.
However, the present offshore sailing study offers valuable information on the redox state, renal function, and motion sickness response during this high demanding activity. OOS has been shown to induce an increase in oxidative stress biomarkers and NO metabolites. A correlation was found also between the increase in NO metabolites level and motion sickness intensity evaluated through questionnaires and symptoms. In this experiment, a transient reduction in renal function was found. Moreover, salivary cortisol increased in response to physical activity and stress induced by navigation. Future studies are required to investigate the biochemical processes and the clinical correlations consequent to maritime exposure.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.