INTRODUCTION:

Chronic exposures to tobacco or its derivatives in various ways are well known to cause adverse effects on health.¹The liver metabolizes the nicotine of the tobacco and detoxifies it.²

Tobacco consumption or exposure to tobacco derivatives is enormous among the population. Bidi is one of the forms of tobacco exposure, being very popular in some of the Asian countries.

'Bidis', 'beedis' or 'beeris' are slim, hand-rolled, unfiltered cigarettes. A bidi consists of about 0.2 grams of sun-dried and processed tobacco flakes, rolled in a tendu leaf (Diospyros elanoxylon) or temburni leaf. The tobacco rolled in bidi is different from that used in cigarettes and is referred to as 'bidi tobacco’.³ Bidis are known as the 'poor man's cigarettes, as they are smaller and cheaper than cigarettes. Bidi making originated in the tribal habit of rolling Sal leaves for smoking.⁴

Inadequate awareness about the dangers of smoking, combined with the addictive potential of bidi smoking, the low price, and perception of people that it is a herbal product with no health risk have increased its popularity.^5,6Studies have shown that bidis are as harmful as or potentially more harmful than manufactured cigarettes.^7,8,9Bidi smoking adversely affects the health of consumers, in terms of early mortality, lung diseases, heart and circulatory diseases, cancers, and oral diseases.¹⁰

Women’s labor constitutes around 44% of total labor employed in bidi tobacco cultivation and about 76% in bidi manufacture.¹¹ Women may roll bidis with infants on their laps, so both mother and child are smeared with tobacco and breathing in the tobacco dust. Since bidi rolling takes place mostly at home, it has an adverse impact on the health of the women workers and other family members, especially children. Bidi rollers, bidi leaves processing, and agricultural workers are exposed to tobacco dust and volatile components of tobacco in their work environment which enters their body via cutaneous and nasopharyngeal routes.

Several studies have reported that smoking is an independent risk factor for the development of non-alcoholic fatty liver disease [NAFLD].^12,13NAFLD is a histologic spectrum of pathologic changes ranging from hepatic steatosis to nonalcoholic steatohepatitis (NASH), an inflammatory phenotype with hepatocyte injury, with or without fibrosis, which can progress to cirrhosis and complications of liver failure and hepatocellular carcinoma.¹⁴NAFLD is one of the most common chronic liver diseases, but the effect of tobacco smoking on the progression of NAFLD is controversial. A recent meta-analytic study demonstrates that smoking is significantly associated with NAFLD, and passive smoking increases the risk of NAFLD about 1.38-fold.¹⁵

Although, traditional drugs are known to have hepatoprotective properties,¹⁶ studies show that liver plays a vital role in endogenous synthesis of cell membrane phospholipids [phosphatidylcholine, phosphatidylethanolamine] and other signaling molecules that are essential during the development and repair of cells. One of them is choline, an essential nutrient obtained through both dietary intake from rich sources like egg yolks and animal sources of protein and also endogenously synthesized involving methyl-group metabolism, particularly in the liver.¹⁷Hepatic homeostasis of phosphatidylcholine is achieved by balancing phosphatidylcholine /choline/ betaine uptake and anabolism [70% from CDP-choline pathway that requires dietary choline and 30% de novo synthesis from phosphatidylethanolamine N-methyltransferase (PEMT) pathway] with phosphatidylcholine catabolism and secretion.¹⁸Choline-deficient diets are known to induce NAFLD in animals. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated, essential fatty acid. There are few plant sources such as corn, olive, palm, soybean, sunflower, canola, walnuts, flaxseed, rapeseed, poppy seed and wheat germ in which it is found in the form of α–linoleic acid (α–LA).¹⁹Converting α–LA into DHA is a variable and inefficient process.²⁰Thus, tissue and circulating DHA levels are primarily determined by their direct dietary intake. DHA-derived products protect the liver from necro-inflammatory injury.²¹Children with NAFLD had improvement from liver steatosis with supplementation of algae DHA.^22,
23

Our previous studies show that combined supplementation of choline and DHA have a synergistic effect in enhancing neurodevelopment and mitigating neurodegeneration by attenuating levels of brain ROS when exposed to stress or cerebral ischemia.^24-28

But, studies exploring the effects of chronic exposure to bidi smoke and dust particulate matter on microstructural alterations in liver morphology and the protective effects of combined dietary choline and DHA supplements on tobacco particulate induced liver damage are largely unknown. We hypothesize that post-weaned pups from Wistar rat dams chronically exposed to bidi smoke and dust particulate matter would have detrimental microstructural alterations in their hepatocytic morphology. We further rationalize that these hepatocytic microstructural non – alcoholic fatty liver alterations can be ameliorated by combined supplementation of choline and DHA to these rat dams.

METHODS:

Animals:

Adult male and female Wistar rats were bred in the animal house. All rats were housed in polypropylene cages containing sterile paddy husk (procured locally) as bedding, throughout the study and maintained under standard conditions with temperature (22–24⁰C), 12 h light/12 h dark cycle and relative air humidity 40–60%. The animals were acclimatized to the laboratory conditions for one week before the start of the experiment. The animals were given a normal pellet diet and water ad libitum. The experiment was conducted according to the ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India, and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines after approval of the experimental protocol by the Institutional Animal Ethics Committee.

Mating of rats:

Female Wistar rats were allowed to mate with one fertile sexually active male overnight. The next day, females were separated, and vaginal smears were taken to detect the presence of sperm for the confirmation of pregnancy and the rat dams were designated as day 0 of pregnancy.

Animal groups:

Rat dams at day 0 of pregnancy were divided into 4 groups [n=6 dams /group].

Group 1: Normal Control group (NC): Rat dams remained in a home cage under standard laboratory conditions.

Group 2: Bidi Smoke exposure group (BS): Rat dams were exposed to bidi smoke in a glass chamber for 1 h/day.

Group 3: Tendu Leaf smoke exposure group (TLS): Rat dams were exposed to tendu leaf smoke in a glass chamber for 1 h/day.

Group 4: Bidi Tobacco dust exposure group (BTD): Tobacco flakes were sprinkled on the floor of the home cage of rat dams along with husk (200g/kg bw).

Group 5: Bidi smoke exposure treated with choline and DHA (CDHA): Rat dams treated with choline and DHA were exposed to bidi smoke in a glass chamber for 1 hr/day

The exposure of the experimental group of rat dams to the appropriate protocols as mentioned above was done during the gestational and lactation period- from day 0 of pregnancy till the end of lactation.

Smoke exposure:

BS (group 2), TLS (group 3), and CDHA (group 5) rats were exposed to smoke in a glass chamber with sufficient ventilation and light. Approximately 50 bidis or 75 tendu leaves were burnt for 1 hr. in a separate glass chamber using an Electric burner machine. With the help of a tube, the smoke was transferred to the chamber where the rat dams were housed.

Choline and DHA:

Choline was supplemented at 4.6 mmol/kg/bodyweight [BW]/day) and DHA (300 mg/kg/day) via oral gavage.

Histological procedure:

At the end of the experimental period, 6 pups from each group (1 pup from each rat dam/group) together with rat dams were sacrificed by overdose of ketamine (IP). The entire liver organ was dissected out and fixed immediately in 10% formalin. After adequate fixation, a further histological process was performed and paraffin sections were prepared. Series of 5µ thick sections were taken using a rotatory microtome and stained with routine Hematoxylin and Eosin stain. A detailed histological study was performed under a light microscope.

RESULTS:

Histological features of Hematoxylin and Eosin-stained liver sections revealed normal hepatocytic morphology together with non-pathological vascular morphology in the livers from normal control group of both rat mothers and their pups (Figure 1).

Figure 1: Representative photomicrographs of liver histology (H&E stained; x40 magnification) from Beedi smoke-exposed group of mother rats (BSM) and their pups (BSP) compared with the same from normal control mother rats (NCM) and their pups (NCP). Mild degree of fatty changes were observed in the background of normal liver histological features in beedi smoke-exposed groups.

The liver microarchitecture of bidi smoke group (group 2) and tendu leaf smoke (group 3) animals presented focal mild fatty changes (Figure 2 and Figure 3).

Figure 2: Representative photomicrographs of liver histology (H&E stained;x40 magnification) from Tendu leaf smoke-exposed group of mother rats (TLSM) and their pups (TLSP) compared with the same from normal control mother rats (NCM) and their pups (NCP). Hepatocytes with mild fatty changes are observed in tendu leaf smoke-exposed groups.

Figure 3: Representative photomicrographs of liver histology (H&E stained;x40 magnification) from Tobacco dust smoke-exposed group of mother rats (TDSM) and their pups (TDSP) compared with the same from normal control mother rats (NCM) and their pups (NCP). Larger vacuoles in the histological sections are evidence of moderate fatty changes in liver hepatocytes of tobacco dust smoke groups.

But the liver section of animals [both mother and their pups] exposed to bidi tobacco dust (group 4) were observed to have moderate fatty changes. Many hepatocytes containing several small, well-defined vacuoles were apparent in the liver microarchitecture (Figure 4).

Figure 4: Representative photomicrographs of liver histology (H&E stained; x40 magnification) from beedi smoke-exposed group of mother rats (BSM) and their pups (BSP) compared with the same from mother rats and pups treated with choline and DHA (CDHAM and CDHAP). Reduced microvesicular fatty changes can be observed in hepatocytes of liver sections from mother rats and pups treated with choline and DHA as compared to the same from bidi smoke (BS) exposed group of rats.

Whereas liver sections of animal groups exposed to bidi tobacco smoke and supplemented choline and DHA [CDHA] were observed to have significantly reduced micro-vesicular and reduced focal mild fatty changes (Figure 5) as compared to the same in age-matched bidi tobacco smoke-exposed rat liver histology.

Figure 5: Representative photomicrograph of liver histology (H&E stained;x 100 magnification) showing hepatocytes from Bidi smoke-exposed rat pups treated with CDHA, showing restoration of normal microarchitecture with reduced microvesicular fatty changes.

DISCUSSION:

Non-alcoholic fatty liver disease (NAFLD) is an obesity-related health complication. NAFLD is generally characterized by the accumulation of lipid (mainly triglycerols) in non-alcoholic individuals.²⁹Exposure to ambient air particulate matter resulting in non-alcoholic fatty liver disease has been reported.³⁰Studies show that, smokeless tobacco users manifest damage to antioxidant defense mechanisms^.31,32 Second-hand smoke has a role in the genesis and progression of metabolic liver disease through the deregulation of genes, molecular pathways, and functional networks involved in lipid homeostasis.³³Results of the present study also corroborate similar findings in rat dams and pups exposed to bidi tobacco smoke with evident hepatocytic fatty changes in their liver histology possibly due to deregulation in lipid homeostasis initiating metabolic liver disease.

Besides epidemiological observations, there is also experimental evidence showing that bidi smoke has an effect that is equally as harmful as cigarette smoke on parameters such as pulse rate, blood pressure, platelet aggregation time, and serum-free fatty acid levels known to be related to the pathogenesis of coronary heart disease and cancers^.34-37 The liver enzymes namely Aspartate transaminase (AST), Alanine transaminase (ALT), Alkaline phosphatase (ALP), and Gamma Glutamyl Transferase (GGT) in smokeless tobacco users are significantly higher than healthy individuals, and serum Malondialdehyde (MDA) level significantly higher in smokeless tobacco chewers than healthy individuals.³⁸It has also been observed to decline antioxidant enzymes, superoxide dismutase (SOD), and catalase activity.³⁹

Avti et al., in their experimental study on rats following the administration of aqueous extract of smokeless tobacco reported histopathological changes in the liver parenchyma showing inflammation and biochemical alteration in the levels of liver antioxidant enzyme status.⁴⁰Petro et al., suggested that increased production of pro-inflammatory cytokines might have been associated with liver cell injury.⁴¹

Studies have reported close relation of waist circumference of tobacco smokers to their visceral adipose tissue distribution which in turn is influenced by serum cortisol level,⁴²low estrogen and testosterone levels.⁴³This altered sex hormone concentration is associated with visceral obesity and metabolic syndrome through its influence on the facilitation of lipolysis and inhibits fatty acid formation.⁴⁴

In patients with unexplained persistent abnormal liver function tests, NAFLD is the commonest histological diagnosis.⁴⁵Studies have emphasized that, when the metabolic syndrome is prevalent, NAFLD might cause chronic liver disease.⁴⁶NAFLD is characterized by steatosis, which can further progress into inflammation, fibrosis and steatohepatitis.⁴⁷Stephanie and Amon reported a rising association between tobacco consumption through cigarette smoking and the advancement of fibrotic liver diseases such as NAFLD.⁴⁸

The passive tobacco particulate exposure to Wistar rats as done in the present study, has not only shown the varying degrees of fatty changes in liver histological sections of rat dams but also showed altered microstructural changes in liver morphology of their post-weaned pups.

Studies indicate that choline is an essential nutrient important for the normal function of all cells. Choline can be obtained both from dietary sources and de novo synthesis in the liver. Moreover, it is known that choline requirement can be met endogenously by a reaction involving phosphatidylethanolamine N-methyltransferase (PEMT) in the liver. Studies show that PEMT transcription increases in a dose-dependent manner in primary mouse and human hepatocytes treated with 17-β-estradiol associated with an increase in protein expression and enzyme activity.⁴⁹Likewise, the PEMT gene is known to be activated by estrogen and aberrant estrogen regulation of the PEMT gene results in choline deficiency-associated liver dysfunction.⁵⁰Dietary choline deficiency in most adult men and postmenopausal women caused the development of signs of organ dysfunction (fatty liver or muscle cell damage). Whereas in premenopausal women, estrogen is enough to activate the expression of the PEMT gene, which allows the endogenous synthesis of required choline in the liver.⁵¹Low choline causes mitochondrial dysfunction which can lead to hepato-steatosis and liver cell death along with the pathogenesis of NAFLD.⁽⁵²⁾An aggressive variety of NAFLD known as non-alcoholic steatohepatitis presents the histological features of higher prevalence of hepatic steatosis and inflammation associated with hepatocyte damage irrespective of liver fibrosis.⁵³Supplementation of choline can minimize or reverse the conditions that mimic the development of NALFD.

DHA is also an essential polyunsaturated omega 3 fatty acid, that has to be consumed in the diet.⁵⁴DHA has hepato-protective action as studies show that its use in treatment reversed histopathological changes of nonalcoholic steatohepatitis (NASH) and lowered the risk of liver lipid peroxidation and improved liver glutathione and total thiol groups concentrations that may prevent progression into advanced stages of NAFLD. The protective effects of omega-3 fatty acids are due to eicosanoid inhibition along with the formation of resolvins and protectins with insulin-sensitizing, antisteatotic and adiponectin inducer effects.⁵⁵DHA supplementation was observed to be associated with anti-oxidative and anti-inflammatory potential in bile duct ligated male Sprague-Dawley rats.⁵⁶

But the synergistic effects of supplementing both essential nutrients choline and DHA in ameliorating NAFLD is largely unknown. Our earlier studies showed that combined supplementation of dietary choline and DHA have a synergistic effect when given during pregnancy by enhancing fetal neurodevelopment²⁴ and when provided to perinatal stressed caused persistent attenuation in ROS or postnatal obese rats mitigated adverse effects on body mass index, serum lipid profile and arterial wall structure respectively, through aging.^26,57Additionally, combined supplementation of dietary choline and DHA to postnatal obese rats or rat models of chronic cerebral hypoperfusion ischemic brain injury also attenuated hippocampal neural cell degeneration and memory deficits. ^{27, 28}

Results of the present study indicate that rat dams and pups exposed to bidi tobacco smoke and supplemented both choline and DHA (CDHA) had a better synergistic effect in significantly ameliorating micro-vesicular and focal mild fatty changes in their liver hepatocytes compared to the same in age-matched bidi tobacco smoke-exposed rats.

CONCLUSION:

Chronic exposure to tobacco particulate matter not only causes cardiovascular, respiratory system disorders and causes cancers, but also affects the metabolic activities of the liver and results in fatty changes. These non-alcoholic fatty liver changes can be effectively ameliorated by combined supplementation of dietary choline and DHA.

In this context, it is to be noted that, tobacco abuse is a chief contributor to morbidity and mortality globally. The mortality rates among bidi smokers are reported to be significantly higher compared to smokeless tobacco users and the same in women bidi workers and their kids exposed to bidi tobacco particulate matter is largely unreported.

ABBREVIATIONS:

CDHA- Choline and docosahexaenoic acid; CDHAM - Choline and docosahexaenoic acid treated mother rats; CDHAP- Choline and docosahexaenoic acid treated rat pups; NC- Unexposed Normal Control; NCM-normal control mother rats; NCP- normal control pups; BS -Bidi Smoke exposure group; BSM-Beedi smoke-exposed mother rats; BSP - Beedi smoke-exposed rat pups; TLS-Tendu Leaf smoke exposure group; TLSM-Tendu leaf smoke-exposed mother rats; TLSP-Tendu leaf smoke-exposed rat pups; BTD-Bidi Tobacco dust exposure group; TDSM- Tobacco dust smoke-exposed group of mother rats; TDSP-Tobacco dust smoke-exposed group of rat pups; NAFLD-Non-alcoholic fatty liver disease; NASH-nonalcoholic steatohepatitis; PEMT- phosphatidylethanolamine N-methyl transferase; H-E-Hematoxylin and Eosin-stained; ROS- Reactive oxygen species

ACKNOWLEDGMENTS:

We gratefully acknowledge our thanks to the Manipal Academy of Higher Education for providing and facilitating all necessary support for this study.

AUTHORS’ CONTRIBUTIONS:

JP and KSR designed the study. JP was responsible for data collection and data analysis, with JP and NK equally contributing to interpret the data, as well as drafting the manuscript for this study, with help from SRS. AB did the liver histological analysis work-up. MKGR and KSR supervised the study and provided critical revisions for the important intellectual content in the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent:

This study was conducted in accordance with the ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India, and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines after obtaining approval from the Institutional Animal Ethics Committee.

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Received on 10.02.2022 Modified on 23.06.2022

Research J. Pharm. and Tech 2023; 16(8):3787-3793.

DOI: 10.52711/0974-360X.2023.00625