Preliminary study on the effect of ozone exposure on blood glucose level in rats

1.Introduction

In recent years, people have paid increasing attention to the health hazards caused by O3 exposure, which is becoming a prominent problem affecting human health following fine particulate matter (PM). In 2021, the WHO revised its Global Air Quality Guidelines based on evidence of health effects associated with long-term O3 concentrations and total and respiratory mortality, adding the seasonal average of peak O3 concentrations (i.e., warm season peaks) with a guideline value of 0.03 ppm [1]. China has strict regulations on indoor and outdoor O3 concentrations. The outdoor O3 standard is that of the Ambient Air Quality Standard [2] in which the maximum concentration of O3 is 0.08 ppm (1 h mean, Class I) and 0.01 ppm (1 h mean, Class II). The indoor O3 standard is that of the Indoor Air Quality Standard in which the maximum concentration of O3 is 0.08 ppm (1 h mean) [3]. As the Chinese economy has grown and its level of workplace automation has increased, many occupational practitioners have received high exposure to O3, which has become a major occupational hazard factor; for example, laser printers emit O3 and volatile organic compounds (VOCs) during operation [4]. Pan [5] found that the levels of O3 and VOCs produced by printers increased significantly during extended operation. Wang et al. [6] found that the average concentration of O3 was 0.173 mg/m3 at the peak of office printing and photocopying work (2 h), which is higher than the Sanitary Standard for Ozone in Indoor Air. Feng et al. [7] investigated and studied the chemical hazards to which the staff of horse racing enterprises in the specified animal disease-free zone were exposed. After detecting the harmful chemical substances in the atmosphere under different working conditions, they found that the maximum exposure concentration of O3 in the air under welding maintenance conditions was 0.51–0.64 mg/m3, which exceeded the maximum allowable concentration (0.3 mg/m3) by nearly twice, so people engaged in welding might suffer from a minor hazard risk, that is, the concentration of O3 exceeds its maximum allowable concentration. Comprehensive waste treatment plants (CWTPs) form a large number of O3 precursors, such as VOCs and nitrogen oxide, resulting in a net accumulation of O3 [8]. Guo et al. [9] conducted an on-site investigation and evaluation of occupational health in a metal component factory. At some work posts, the O3 exposure exceeded the national standard concentration and became an important factor of occupational hazards.

With the deepening of research on the harm caused to the body by O3 exposure, it has been found that O3 not only causes serious damage to the lungs and cardiovascular system but also has a certain impact on blood glucose levels, which has drawn the attention of environmental biology experts. Diabetes is the major cause of the incidence rate and mortality of noncommunicable diseases. In 2012, the WHO reported that the global adult prevalence rate had risen to 8.5%, causing 1.5 million deaths. O3 exposure is considered a potential main pathogenic factors for the onset of Type 2 diabetes (T2DM). Epidemiological studies have shown that O3 exposure can aggravate the increase of insulin resistance (IR) [10] and may increase the risk of T2DM [11]. Paul et al. [12] conducted a cohort analysis of 4.8 million Canadian adults without a history of diabetes for more than 10 years. The results showed that the average O3 concentration at the 3-year baseline was 46.5 ppb, and each 6.3 ppb increase in O3 concentration was associated with 0.5% increased risk of T2DM events. Hernandez [13] showed that an increase of 10 ppb in average annual O3 concentration from 2002 to 2008 was associated with a 6% increase in the hazard of T2DM events in the United States. Liu [14] conducted a cohort study on individual O3 exposure among more than 30,000 rural people in Henan Province. For every 4.04 μg/m3 increase in O3 exposure, the prevalence of T2DM and fasting blood glucose (FBG) levels increased by 53.3% and 0.292 μmol/L, respectively. Elderly people and men are vulnerable to the effects of O3 exposure on T2DM and FBG. However, the effect of O3 exposure on blood glucose level and its inducing mechanism is not yet fully understood. The above study was conducted on the relationship between chronic O3 exposure and T2DM. However, the impact of acute O3 exposure on blood glucose level has been given less attention. In real life, the duration of severe O3 pollution in the environment is short, and workers at posts with severe O3 exposure are highly mobile. Therefore, we need to pay more attention to the impact of acute O3 exposure on blood glucose metabolism. This experiment explored the effects of acute O3 exposure at various times and concentrations on the change of blood glucose levels using an exposure model test with rats, thereby providing references for the inducement and prevention of diabetes.

2.Materials and methods

3.Results

3.2Fasting blood glucose and random blood glucose results

FBG detection results are shown in Fig. 1A. Compared with the control group, the FBG detection values at 1 day, 3 days and 6 days after exposure to 0.1 ppm concentration had no statistical significance. At a concentration of 0.5 ppm, the blood glucose value on the sixth day was significantly higher than that of the control group (P= 0.037). At a concentration of 1.0 ppm, the blood glucose values on the third and sixth days were significantly higher than those in the control group (P= 0.039, 0.02). RBG was detected in rats under normal diet and water intake. The specific results are shown in Fig. 1B. At a concentration of 0.1 ppm, the blood glucose values on the third and sixth days were substantially higher than those in the control group (P= 0.015, P< 0.001). The blood glucose value on the sixth day was significantly higher than that on the 1st day (P= 0.013). At a concentration of 0.5 ppm, the blood glucose values on the third and sixth days were significantly higher than those in the control group (P= 0.002, P< 0.001), considerably higher than those on the first day (P= 0.046, P< 0.001), and the blood glucose values on the sixth day were significantly lower than those on the third day (P= 0.032). At a concentration of 1.0 ppm, the blood glucose values on the third and sixth days were significantly higher than those in the control group (P= 0.001, P< 0.001), and the blood glucose values on the third and sixth days were significantly higher than those on the first day (P= 0.006, 0.001)

Figure 1.

FBG and RBG values at 0.1 ppm, 0.5 ppm, and 1.0 ppm O3 exposure for 1, 3 and, 6 days. The values in the bar graphs are shown as mean ± SE (n= 10); P*< 0.05 compared with the control group, P**< 0.01 compared with the control group, P#< 0.05 compared with the blood glucose value on the first day, P#⁢#< 0.01 compared with the blood glucose value on the first day, PΔ< 0.05 compared with the blood glucose value on the third day and PΔ⁢Δ< 0.01 compared with the blood glucose value on the third day.

Figure 2.

GTT values at 0.1 ppm, 0.5 ppm, and 1.0 ppm O3 exposure for 1, 3 and, 6 days. The values in the graphs are shown as mean ± SE (n= 10); P*< 0.05 compared with the control group, P**< 0.01 compared with the control group, P#< 0.05 compared with the 0.1 ppm concentration group, P#⁢#< 0.01 compared with the 0.1 ppm concentration group, PΔ< 0.05 compared with the 0.5 ppm concentration group and PΔ⁢Δ< 0.01 compared with the 0.5 ppm concentration group.

4.Discussion

In this experiment, rats were exposed to three different O3 concentrations for different duration, causing different effects on the FBG, RBG, and GTT values in rats. These blood glucose values can serve as important reference data for clinical diagnosis of T2DM, especially GTT 120 min and FBG. The increase in FBG can be taken as a sensitive indicator of pre-diabetes and general diabetes [17]. In humans, GTT is used in toxicological studies to assess the ability of the regulatory mechanisms to determine blood glucose concentrations, providing a physiological overview of changes in glucose tolerance [18]. The results of this experiment showed that O3 effected all the above blood glucose values. The blood glucose used in the experiment was rat tail venous blood detected by a rapid blood glucose detector. The experiment proved that there was no difference in the blood glucose value of rats detected by a rapid blood glucose meter and a biochemical meter [19]. The global prevalence of T2DM has risen dramatically in recent decades, with the International Diabetes Federation estimating that at least 415 million people aged 20 and 79 years had diabetes in 2015. That number is expected to rise to 642 million by 2040. China also has the largest number of diabetics in the world [20]. The etiology and pathogenesis of T2DM are not yet fully understood. Epidemiological and environmental toxicological studies have shown that O3 exposure can increase IR and the prevalence of T2DM. Research has also shown that short-term O3 exposure is positively correlated with FBG, insulin, HOMA-IR, and islet β-cell secretory function index (HOMA-B) [17]. Bass et al. [15] exposed rats of 1, 4, 12 and, 24 months to 0.25 ppm and 1.0 ppm O3, the experiment showed that acute O3 exposure in rats of all months of age resulted in increased FBG and decreased glucose tolerance. Liu et al. [14] found that the increase in O3 exposure concentration was positively correlated with the increase of FBG level and risk of T2DM. When the mixed exposure effects of PM2.5, PM10, or NO2 were constructed in a dual-pollution model and combined with the region, temperature and relative humidity, the results of O3 exposure and T2DM and FBG were basically unchanged. These results indicate that O3 exposure as an independent factor effects blood glucose metabolism. Desinia [21] exposed Wistar rats to 0.25, 0.5, and 1.0 ppm O3 for GTT over two days. Both the 0.5 and 1.0 ppm test groups showed elevated blood glucose and abnormal blood glucose tolerance, while the 0.25 ppm group did not experience hyperglycemia or abnormal blood glucose tolerance. Some studies have shown that no significant changes in glucose tolerance or FBG were observed in any study group when exposed to 0.25 ppm O3 [18]. In our experiment, the 120 min mean values of FBG, RBG and GTT were significantly increased on different days across the three concentration exposure groups. Although they did not exceed the Wistar rat model T2DM venous blood glucose limit of more than 16.7 mmol/l, they exceeded the normal value of rat venous blood glucose (5.8 ± 0.5 mmol/l) [19]. In addition, the higher the concentration, the more obvious the increase in blood glucose value. It can be seen that the effects of acute O3 exposure on blood glucose are concentration-dependent. Some scholars have carried out relevant research, showing that the effects of O3 exposure below 0.25 ppm on the blood glucose of rats are mild, but impaired glucose tolerance can be observed after long-term low-concentration O3 exposure [22]. The experimental results of this paper show that the RBG and FBG levels of Wistar rats exposed to 0.1 ppm, 0.5 ppm and, 1.0 ppm concentrations for 1 day, 3 days and 6 days were significantly higher than those of the control group, and increased with the extension of the exposure time. However, the blood glucose values of GTT at the concentration of 0.1 ppm for 1 day, 3 days and 6 days did not significantly increase compared with the control group, and the blood glucose values of GTT at 90 min and 120 min on each exposure day at the concentration of 0.5 ppm did not significantly increase compared with the control group. The three exposure concentrations in this experiment can cause significant increases in FBG and RBG, and only the change in the GTT blood glucose value of Wistar rats at 1 ppm exposure concentration was statistically significant.

The mechanism of O3 exposure causing changes in blood glucose levels and T2DM is still not fully understood. Many researchers have proposed a number of possible pathways, chiefly pulmonary and systemic inflammatory reactions. After exposure to environmental pollution, the release of inflammatory mediators from the lungs into the circulatory system increases [23]. O3 exposure can cause changes in inflammatory factors and lung tissue injury [24]. Studies have shown that tumor necrosis factor (TNF)-α and Interleukin (IL)-6 enter the systemic circulation and act on target tissues or organs, mainly the liver, muscles, and fat, which are sensitive to insulin [25]. Inflammatory factors are widely involved in the regulation of blood glucose, lipid and energy metabolism in the body, causing a chronic low-grade inflammatory reaction that leads to the damage of the insulin signaling pathway and the decrease of the body’s glucose utilization efficiency, in turn leading to the occurrence of IR [26]. Among the various possible causes of elevated blood glucose and IR induced by O3, most researchers believe that inflammatory reaction/lung injury may be the center of the biological mechanism associated with elevated blood glucose induced by O3 [27]. Ying et al. [28] exposed KKAy rats to O3 at 0.5 ppm for 13 consecutive working days, and the results confirmed that plasma TNF-A as well as the expression of VCAM-1, iNOS, and IL-6 in lungs and adipose tissue were increased; the number of proinflammatory CD11 macrophages was increased by 200% in adipose tissue, and fasting plasma insulin level and HOMA-IR were significantly decreased. Second, O3 induces oxidative stress and endoplasmic reticulum stress to increase the body’s blood glucose level. Vella et al. [29] exposed rats to O3 at a concentration of 0.8 ppm and found that the concentrations of FBG and insulin in rats were significantly increased; the concentrations of oxidative stress biomarkers 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE) and malondialdehyde (MDA) in the blood and bronchoalveolar lavage fluid (BALF) of the rats were increased; the concentrations of protein carbonyl groups in lungs was increased; the ratio of GSH/GSSG in the red blood cells was decreased; and the expression of the endoplasmic reticulum stress protein gene in the muscle was increased. O3 exposure induces a rise in blood glucose caused by nerve reflexes in the lungs and brain, and activates the stress response regions in the nucleus tractus lonsus (NTS) and hypothalamus by stimulating the C fibers of the pulmonary vagus nerve [30]. Acute O3 exposure increases the level of stress hormones in the circulation, such as adrenaline and corticosterone [31, 21]. O3 exposure also leads to cardiac autonomic effects, hypothermia, and bradycardia in rats [32], as well as hyperglycemia, glucose tolerance and systemic changes in circulating metabolites related to peripheral glucose, fat, and amino acid metabolism in rats [21]. These changes reflect classic stress-mediated changes in homeostasis, including the activation of sympathetic neurons and the hypothalamic-pituitary-adrenal (HPA) axis [21]. Acute O3 exposure increases the serum corticosterone of rats, which is a sign of HPA axis activation. Miller [33] recruited 24 volunteers to expose them to 0.3 ppm concentration of O3. The research showed that the serum samples collected after O3 exposure showed significantly increased cortisol and corticosterone compared with the air group. Thomson et al. [31] exposed rats to O3 at concentrations of 0.4 ppm and 0.8 ppm, the results showed that the plasma levels of corticotrophin and glucocorticoid in the plasma were increased, especially for O3 at 0.8 ppm. Bass [15] showed that adrenalin, a neurohumoral regulator of the sympathetic nervous system, was significantly increased in serum on the second day after exposure to 1 ppm O3. Finally, O3 exposure causes changes in lipid metabolism and increases blood glucose. Miller [21] conducted an O3 exposure experiment on Wistar Kyoto rats, showing that O3 exposure can increase the glycolytic cycle metabolite, long-chain free fatty acids, and branched-chain amino acids, and reduce the circulating metabolite of 1,5-anhydrous sugar alcohol, bile acid and tricarboxylic acid (TCA). It can be seen that O3 exposure interferes with the normal glycogen synthesis and glycolytic pathway of the body. The increase of lipid and protein catabolic residual products in the circulation may play a role in peripheral IR. Vella et al. [29] also found that O3 exposure led to the increase of lipid and protein oxidation residual products in the BALF and serum, and these residual products increased muscle IR.

The concentrations of O3 used in this experiment were 0.1 ppm, 0.5 ppm and, 1.0 ppm, which are higher than the allowable occupational health limits stipulated by the state. However, studies have shown that mice need three to four times the amount of O3 to obtain the same amount of O3 as humans [34]. Therefore, the concentration of O3 used in our study to simulate the impact of acute O3 exposure on human blood glucose metabolism basically conforms to the exposure concentration of O3 in some actual jobs and environments. The exposure concentrations of the three groups in this experiment caused the rise of FBG and RBG in the body with the extension of the exposure time. The concentration of 1.0 ppm can cause the rise of the GTT blood glucose value by 120 min on the third and sixth days. O3 exposure has different effects on blood glucose levels, providing data references for further exploring the etiology and pathogenesis of T2MD. To prevent the effects of O3 exposure on the body’s blood glucose level, it is necessary to reduce the time and concentration of occupational O3 exposure, and strengthen exercise activities. Studies have shown that the glucose tolerance of sedentary rats will be impaired after O3 exposure [16]. Physical activity may be effective in reducing the burden of high FBG caused by T2DM and O3 exposure [14].

5.Conclusion

In this experiment, rats were exposed to O3 at different concentrations and times to detect FBG, RBG and GTT. Each exposure concentration and time induced elevated FBG and, RBG, and the exposure time had a cumulative effect on the elevated blood glucose level. At concentrations of 0.5 ppm and 1.0 ppm, the blood glucose of GTT was significantly affected. The results of this experiment provide data to support the hypothesis that O3 exposure increases the blood glucose level of the body. As the prevention of chronic noncommunicable diseases is better than treatment, the impact of our research on public health provides scientific evidence of the potentially harmful effects of O3 air pollution on blood glucose metabolism and the health of people. This experiment lacks the study of O3 on the mechanism of insulin tolerance and elevated blood glucose.