Interplay of Obesity, Ethanol, and Contaminant Mixture on Clinical Profiles of Cardiovascular and Metabolic Diseases: Evidence from an Animal Study

Cardiovascular diseases (CVDs) are the number one cause of death worldwide, with an estimate of 17.9 million loss of lives each year (WHO 2021, https://​www.​who.​int/​health-topics/​cardiovascular-diseases). Atherosclerosis is the main underlying cause of CVDs that is characterized by hypercholesterolemia, hypertriglyceridemia, vascular inflammation, platelet activation, and thrombosis [1]. Dyslipidemia, immune cell activation, and endothelial dysfunction are the key processes involved in the initiation and progression of atherosclerosis [2‐5]. Various biomarkers reflecting changes in these processes have been used in risk stratification, prevention, and treatment of CVDs [6‐12].

Many modifiable and non-modifiable factors including diet, lifestyle, age, and genetic background can contribute to the pathogenesis of CVD. Obesity, either acquired or genetic, is a well-known risk factor for CVD [13, 14]. Non-alcoholic fatty liver disease (NAFLD), associated with obesity, is often linked to CVD outcome [15, 16]. Evidence also supports that heavy chronic alcohol consumption increases the risk of CVDs [17‐20]. Systemic arterial hypertension has a strong, continuous positive association with CVDs [21].

Both experimental and epidemiological studies have demonstrated that environmental contaminants such as heavy metals, polychlorinated biphenyls (PCBs), and organochlorine pesticides are potential risk factors for obesity, diabetes, and CVDs [22‐26], although the underlying mechanisms are not fully understood. Serum PCBs levels were found to be associated with elevated serum triglyceride [27] and cholesterol levels [28], increased blood pressure [29, 30], and higher rates of coronary heart disease and myocardial infarction in exposed populations [31]. Elevated methylmercury (MeHg) exposure was associated with increased diastolic and systolic blood pressure [32‐35]. In experimental animals, MeHg decreased circulating levels of paraoxonase-1 (PON1), the major anti-atherosclerotic component of high-density lipoprotein (HDL) [36]. It also increased serum levels of oxidized low-density lipoprotein (Ox-LDL) that are known to contribute to inflammation and the formation of plaque. In addition, MeHg also increased serum monocyte chemotactic protein-1 (MCP-1), a cytokine known to be involved in monocyte infiltration and atherosclerosis. These effects of MeHg implies an increased risk of CVDs in exposed animals [36].

Some circumpolar populations have an increased prevalence of obesity, hypertension, diabetes, and/or CVD [37‐39]. These chronic diseases have also been associated in the scientific literature with elevated levels of various environmental contaminants, alcohol use, and dietary shift from traditional to non-nutrient-dense foods [40‐42]. However, there is a lack of understanding how multiple risk factors such as ethanol consumption, obesity, and contaminant exposure may interplay on the pathogenic processes and outcomes of these chronic diseases, especially CVDs. In a previous study, we investigated the effects and interactions of obesity, ethanol (EtOH), and a contaminant mixture (CM) on liver cholesterol homeostasis and energy metabolism in male obese and lean JCR rats. The rats were orally exposed to a CM containing 22 organic and inorganic environmental contaminants found in human blood or serum of highly exposed Northern populations at three dose levels, 0, 1.6, or 16 mg CM/kg BW/day, with or without co-exposure to 10% ethanol in drinking water [43]. Our results suggested that obesity in JCR rats was associated with hepatosteatosis, while EtOH exposure worsened the hepatosteatosis associated with obesity [43]. The CM exacerbated the existing hepatosteatosis in the obese rats, with or without co-exposure to EtOH [43].

In this study, we determined the effects of CM, EtOH, and obesity, alone and in combination, on over sixty clinical and physiological parameters related to risks and diagnosis of CMD in JCR rats.

In our previous study, we examined the interaction of CM, EtOH, and obesity on parameters related to liver and pancreas health [43, 44]. In this study, we further determined the interplay of CM, EtOH, and obesity on the clinical biochemistry, physiology, hematology, histology, and some specific biomarkers related to CMD in JCR rats.

In the absence of EtOH and contaminants, the obese JCR rats had mild degree of heart histological lesions with focal cardiomyocyte necrosis and inflammatory cell infiltration that were similar to what was described for myocardial infarction in humans [47], which were not observed in the lean JCR rats. These obese rats were hyperlipidemic with several fold higher serum levels of TG, TC, LDL-C, and HDL-C than the normal lean rats, which is consistent with the typical serum lipid profile associated with obesity in humans [48, 49]. The obese rats also had an elevated systemic inflammation as shown by significantly higher NC, N/L-C, PLT, MPV, and serum CRP, and MCP-1 levels than the lean rats. This is in coherence with the inflammatory status associated with obesity found in humans [50, 51]. In addition, the obese rats had higher RBC and RDW, and lower MCV than the lean rats, which has also been found in humans with higher BMI [52] and waist circumference [52, 53], hypertension [54], insulin resistance [52], obesity and type 2 diabetes (T2D) [55].

The obese rats also displayed low serum creatinine, which may be attributed to low muscle mass as what is found in humans [56‐58]. In fact, serum creatinine was also found to progressively decrease with obesity and T2D in nonhuman primates [59]. The lower serum creatinine levels in the obese rats paralleled with the decrease in serum Cl, but not Na, and the increase in serum UA. Low serum Cl is common in patients with chronic heart failure and is associated with worse outcomes [60, 61]. Cl is known to play an important role in regulating blood pressure [62, 63]. Cl depletion has been shown to stimulate renin secretion in the macula densa resulting in increased systemic blood pressure. UA has been shown to play a crucial role in pathogenesis of hypertension through potentially multiple mechanisms including upregulation of the renin–angiotensin–aldosterone system and increased oxidative stress and inflammation [64] Hyperuricemia was associated with metabolic syndrome [65] and increased risk for hypertension [66]. With the low serum Cl, high serum UA, and high water intake, the obese JCR rats would likely develop hypertension. Interestingly, in parallel with these changes, the obese JCR rats also expressed higher levels of serum 6-keto-PGF1α (hydrolytic product of prostacyclin), and NO, two key vessel dilators [67], which might have been activated as a compensatory mechanism to stabilize blood pressure. The higher circulating levels of 6-keto-PGF1α in the obese rats is consistence with its role in adipogenesis and obesity development [68]. All aforementioned observations provided experimental evidence for an increased risk of CVD associated with obesity.

Changes in serum aminotransferases levels have been found in many disease conditions in humans with NAFLD being most common [69, 70]. The elevated serum ALT, AST, ALP and TP levels in the obese JCR rats is consistent with the presence of the NAFLD observed in our previous study under the same experimental conditions [43], suggesting that hepatosteatosis associated with obesity is accompanied by liver damage, leading to increased serum protein levels and altered protein profiles. Changes in serum protein levels and profiles may affect blood viscosity, resulting in increased cardiovascular risks, as what is found in NAFLD patients [71, 72]. Elevated serum ALT levels have been linked to increased cardiovascular and metabolic risks in humans [73]. Disruption of the intestinal microbiome and the related metabolites are believed to contribute to the onset and progression of NAFLD [74‐76]. Higher plasma EtOH were also found in children with NAFLD, and positively correlated with insulin, leptin, and TG [77]. In ob/ob mice, elevated plasma EtOH was attributed to decreased expression of cytochrome P450 2E1 and alcohol dehydrogenase in the liver as compared to lean control mice [77]. It is intriguing that, in this study, the obese JCR rats with hepatosteatosis had lower serum EtOH than the lean rats, while exposure to EtOH did not affect serum EtOH. It is possible that the obese JCR rats have different microbiome and/or EtOH metabolism from human NAFLD patients and ob/ob mice, although more investigations are warranted to clarify this.

Serum CK activity reflects levels of muscle damage and liver clearance of CK, and has been positively associated with blood pressure in some human populations [78, 79]. Elevated serum CK has been shown to predict first-ever myocardial infarction in Japanese populations [80]. This is coherent with the markedly increased serum CK detected in the obese JCR rats, suggesting that liver and muscle damage associated with obesity may contribute to the risk of CVD. Being mainly expressed in pancreas and liver, GK plays a primary role in regulating blood glucose homeostasis through affecting insulin release from pancreatic cells, and glycogen synthesis [81] and lipogenesis [82] in hepatocytes. Compromised liver and β-cell GK function and expression has been associated with patients with T2D, and GK activation has been shown to be effective in treating T2D [82]. Elevated GK activity in the obese JCR rats might prevent hyperglycemia, but would increase lipogenesis and lipid accumulation in the liver [43]. LDH is highly expressed and plays a major role in the production of lactate during anaerobic glycolysis in adipose tissue. Lactate released from adipose tissue is the main substrate for lipogenesis in the liver as a way to control glycemia in response to excess glucose level in the circulation [83]. Increases in serum ALT and LDH levels paralleled the increases in the hepatic ratios of alanine/pyruvate and lactate/pyruvate, respectively, in a rat model of NAFLD, suggesting their involvement in NAFLD [84]. The elevated serum LDH in the obese JCR rats is consistent with its positive correlation with obesity and fatty liver found in humans [85, 86].

In this study, the obese JCR rats had significantly higher tHg concentrations in serum, liver, and kidney than the lean rats, when they were exposed to background contaminant levels present in the vehicle group. When they were exposed to the low-dose CM, the obese rats accumulated higher levels of total mercury in all tissues examined than the lean rats. This could be attributed to both higher intake of mercury from food and water, and lower detoxification and excretion capability of the obese than the lean rats, suggesting that obese individuals may be prone to higher mercury accumulation. This is consistent with the reported link between mercury exposure, obesity and obesity related clinical profile in some human populations [87‐90]. However, it remains to be established if mercury exposure caused obesity and related health effects, or whether obesity and related health deficits resulted in higher retention of mercury. Obesity not only affected tissue mercury levels, but also altered tissue distribution of mercury, rendering higher proportion of mercury distributed in the liver and less in kidney, pancreas, and brain tissues, when compared to lean rats. This could be attributed to the increased fat accumulation in the liver of obese rats.

The results of this and our previous studies suggest that obesity has a broad impact on the physiological- and organ-specific parameters. In contrast, the influence of EtOH on these parameters is more specific and different in lean from obese rats. In our previous study [43], we found that in the obese rats, EtOH increased hepatic expression of geranylgeranyl diphosphate synthase and diphosphomevalonate decarboxylase involved in cholesterol synthesis [43]. It also increased hepatic expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1), an enzyme known to be involved in EtOH metabolism and oxidative stress, and elevated in obesity and hepatosteatosis in humans [91]. In addition, EtOH decreased hepatic expression of ATP5A, a subunit of mitochondrial ATP synthase involved in mitochondrial ATP production [43]. These changes in liver observed from our previous study paralleled with decreases in serum TG, TC, LDL-C, BUN, UA, TP, and ALP observed in this study, suggesting that EtOH exposure disrupted hepatic mitochondrial energy production, lipid metabolism, urea synthesis and/or secretion, and metabolism of purine bases, complicating clinical profiles and diagnosis. Although the decreased serum TG, TC, and LDL-C by EtOH exposure may lessen the risk for atherosclerosis in the obese rats, the decreased serum 6-keto-PGF1α and NO in the obese rats exposed to EtOH may compromise the capability of obese rats to regulate blood pressure, increasing the risk for hypertension.

The effects of CM in obese rats were mostly suppressive and/or damaging. In our previous studies [43], we found that obesity in JCR rats were associated with hepatosteatosis, which was exacerbated by exposure to ethanol and/or CM. While ethanol increased protein levels of some enzymes involved in cholesterol synthesis in the liver, CM decreased the levels of the same and other enzymes involved in cholesterol synthesis in the presence and absence of ethanol, suggesting that CM exposure counteracted on the action of ethanol in the liver. CM, but not ethanol, decreased expression of proteins involved in lipid-binding (liver fatty acid binding protein, LFABP) and transport (ATP-binding cassette transporter A1, ABCA1) in the liver, which paralleled with decreases in serum TG, TC, LDL-C and HDL-C by CM exposure observed in this study. This suggests that CM exacerbated hepatosteatosis by increasing lipid synthesis and/or decreasing lipid and/or lipoprotein export from liver to circulation. This also suggest that different mechanisms may be involved in the effects of ethanol from those of CM on hepatosteatosis. The decreased serum lipid and lipoprotein by high-dose CM may relieve the risk for atherosclerosis, however, can also increase the chance for misdiagnosis and overlooking of the hepatosteatosis associated with obesity. The increased serum CRP, an acute response protein release from liver known to be associated with CVD, and the decreased serum PON1, an antioxidant enzyme protecting HDL-C from oxidation, ApoA1, a HDL-C associated protein known to play an role in maintaining HDL-C function, and 6-keto-PGF1α, a non-enzymatic hydrolysis product of prostacyclin that functions as vessel dilator, by high-dose CM in the obese rats suggest a further increase in the risk for CVD and hypertension in these rats. In addition to heart and liver injuries, CM also disrupted pancreatic structure and function, causing decreased α and β cell mass, increased pancreatic iron deposit and monocyte infiltration, and decreased production and release of insulin and glucagon in obese rats [44]. These changes paralleled with elevated serum iron and islet cell antibodies and decreased serum amylase and glutamic acid decarboxylase autoantibody. These results suggest that CM exposure may promote the progression of disease state from features of T2D to features of T1D in the obese rats. The most intriguing finding of this study was that both low- and high-dose CM increased serum EtOH levels in both lean and obese rats with or without co-exposure with EtOH. This could be attributed to increased endogenous production of EtOH from microflora or decreased metabolism of EtOH, although more studies are warranted to confirm this. In fact, contaminants have been shown to affect gut microflora, while microflora can also affect metabolism of contaminants [92, 93]. High-alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) was associated with up to 60% of individuals with NAFLD in a Chinese cohort [94]. In this study, CM exacerbated the hepatosteatosis associated with obesity. However, the sources and health consequences resulted from CM-induced increase in endogenous EtOH observed in JCR rats remain to be investigated.

In summary, findings from our previous and current studies suggested that in JCR rats, obesity was associated with clinical profiles and organ structural and functional changes that points to increased risk for CVD (Fig. 7). Obesity also increased the risk for contaminant-induced toxicity. Heavy exposure to EtOH exacerbated the state of NAFLD and hyperinsulinemia, while lessening the state of hyperlipidemia associated with obesity. In addition, EtOH exposure increased the risks of developing hypertension and liver intoxication by contaminants in obese rats. Heavy exposure to CM lessened or diminished hyperlipidemia and hyperinsulinemia, but intensified the hepatosteatosis associated with obesity. CM exposure worsened systemic inflammation and cardiomyocyte injury, while also increasing the risk for hypertension in obese rats. Combined exposure to EtOH and CM caused increased liver toxicity, and more complicated physiological profile with marker changes pointing to different health outcomes. MeHg may be responsible for the effects of low-dose CM on circulating TG, Cl, PON1, and EtOH levels. Endogenous EtOH production and metabolism may be a sensitive target of CM, which requires further investigation. Our studies demonstrated that JCR rats are very useful animal models for studying the effects of obesity, ethanol and/or exposure to chemical contaminants, and their interplay on biomarkers related human CMD. However, the human implications of our findings may be limited due to species differences in metabolism, nutritional status, and contaminant exposure levels, which should be considered in interpretation of the information generated from JCR rats. The high CM dose group used in this study represents a scenario that one is exposed to all the component chemicals at ten times of the highest level found in human blood of some highly exposed Northern populations, which is unlikely to happen in real world. Nonetheless, it serves as a positive control for studying the potential adverse health effects of CM.