Tobacco Snuff-Induced Hepatic Alterations

MATERIALS AND METHODS

Experimental animals: Experimental animals (42) were purchased from the animal farm of Anthonio Research Center, Ekpoma, Edo State, Nigeria. After this was transferred to the experimental site and allowed 2 weeks of acclimatization. Wooden wire mesh cages were used to house the experimental animals under standard laboratory procedures⁶. The animal acclimatization, substance procurement (tobacco leaves and potash), actual animal experiment and evaluation of results, lasted from October, 2012 to January, 2013. However, the actual administration of tobacco snuff to the experimental animal lasted for 8 weeks.

Ethics on the use of animals in experimental studies: As contained in the US guidelines, the protocols employed were according to our Institutional guidelines as well as internationally accepted practices for the use and care of laboratory animals^6,20.

Substance of the study: Botanically identified tobacco leave (dried) with potash was purchased from a recommended market (Ogbete Main Market, Enugu State, Nigeria). The botanical identification of the tobacco leaves was carried out by a botanist in the Department of Botany at Ambrose Alli University, Ekpoma, Edo State, Nigeria⁶.

Substance preparation: Mortar and iron pestle were used to blend dried tobacco leaves and potash into powder and were stored before the study. To obtain the graded doses of tobacco snuff used in the study, an electronic balance (Denver Company, USA, 200398.IREV. CXP-3000) was employed and feed pellets were prepared as described by Ugbor et al.⁶ and Nwaopara et al.²¹.

Animal grouping: The experiment was divided into four phases of 2 weeks each: 1st phase was for 2 weeks, the second phase was for 4 weeks, the third phase was for 6 weeks and the fourth phase was for 8 weeks. Each test group was divided into four phases representing 2, 4, 6 and 8 weeks duration. The experimental animals were fed with graded doses of tobacco snuff and at the end of each 2, 4, 6 and 8 weeks, respectively, the animals were sacrificed for organ collection⁶.

Study duration: The preliminary studies, animal acclimatization, substance procurement (tobacco leaves and potash), actual animal experiment and evaluation of results, lasted for five months. However, the actual administration of tobacco snuff to the experimental animals lasted for 8 weeks (2, 4, 6 and 8 weeks)⁶.

Substance administration: In the first phase (2 weeks), group A (control) received 100 g of feed and distilled water only whereas, test groups B, C and D received, 97.6 g of feed+2.4 g of tobacco snuff, 95.2 g of feed+4.8 g of tobacco snuff and 92.8 g of feed+7.2 g of tobacco snuff, respectively. Each test group received distilled water given ad libitum.

In the second phase (4 weeks), group A (control) received 75 g of feed and distilled water only, whereas test groups B, C and D received, 72.84 g of feed+2.16 g of tobacco snuff, 70.68 g of feed+4.32 g of tobacco snuff and 68.52 g of feed+6.48 g of tobacco snuff, respectively.

In the third phase (6 weeks), group A (control) received 50 g of feed and distilled water only, whereas test groups B3, C3 and D3 received, 48.56 g of feed+1.44 g of tobacco snuff, 47.12 g of feed+2.88 g of tobacco snuff and 45.68 g of feed+4.32 g of tobacco snuff, respectively.

In the fourth phase (8 weeks), group A (control) received 25 g of feed and distilled water only, whereas test groups B4, C4 and D4 received, 24.28 g of feed+0.72 g of tobacco snuff, 23.56 g of feed+1.44 g of tobacco snuff and 22.84 g of feed+2.16 g of tobacco snuff, respectively.

The concentrations of tobacco used in this study were deduced from the work of Bagchi et al.²² while, that of potash was deduced from Ugbor et al.¹⁰.

Sample collection: At the end of each phase, animals were sacrificed and the liver organs harvested were fixed in 10% formol saline for histological processing.

Histological analysis: Following the Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), Ile-Ife, Osun State, Nigeria processing schedule, the tissues were processed using an automatic tissue processor. The tissue blocks were then trimmed and sectioned serially at 3 mm on a rotary microtome. The sections were floated in the water bath at 55°C and picked up by the use of clean frosted end slides. The frosted end slides were placed on the hot plate for about 40 min for adequate attachment of the sections on the slides after which the sections were dewaxed, hydrated, air-dried and stored in a slide box ready for the staining process. Haematoxylin and eosin were used for routine staining while the tissue slides were mounted with dibutyl phthalate propylene xylene (DPX). Microscopy and micrograph were performed with a light microscope and a digital camera attached to the microscope.

RESULTS

The histological features of the liver tissues as observed under the microscope for the control group (A) and test groups (B, C and D) were represented by the micrographs in Fig. (1-17). The results showed that the liver section from the control group (A) presented normal histological features (Fig. 1a-c) whereas the test groups showed alterations in the normal histological architectures. Specifically in group B, 2 weeks sections presented edematous changes, severe parenchymal erosion, cellular necrosis and vacuolations (Fig. 2-4). Group B, 4 weeks sections presented cellular necrosis, inflammatory cell infiltration with hemorrhagic exudations and fatty changes with vacuolations (Fig. 5). Group B, 6 weeks sections presented edematous changes with hemorrhagic signs and cellular necrosis with vacuolations (Fig. 6). Group B, 8 weeks sections presented degenerative fatty changes with cellular necrosis, inflammatory cell infiltration and vacuolations (Fig. 7).

Fig. 1(a-c):

Control section (Liver, H&E×400) showing normal tissue cytoarchitectural features of the liver

Fig. 2(a-b):

Test group B (2 weeks) sections (Liver, H&E×400) showing edematous changes with parenchymal erosion (a) (triangular arrows) as well as cellular necrosis (b) (encircled)

Fig. 3(a-c):

Test group B (2 weeks) sections (Liver, H&E×400) showing cellular necrosis (encircled), vacuolations (squared)

Fig. 4(a-b):

Test group B (2 weeks) sections (Liver, H&E×400) showing severe parenchymal erosion

Fig. 5(a-d):

Test group B (4 weeks) sections (Liver, H&E×400) showing cellular necrosis (a and b, encircled), inflammatory cell infiltration (squared) with hemorrhagic exudations (line arrow) (c) and fatty changes with vacuolations (triangular arrows) (d)

Fig. 6(a-d):

Test group B (6 weeks) sections (Liver, H&E×400) showing edematous changes with hemorrhagic signs (triangular arrow) and cellular necrosis (encircled) with vacuolations (a, c and d)

Similarly, group C, 2 weeks sections presented cellular necrosis, vacuolations, hemorrhagic exudations and severe haemorrhage (Fig. 8, 9). Group C, 4 weeks sections presented cellular necrosis, vacuolations and severe cellular infiltration with haemorrhage (Fig. 10). Group C, 6 weeks sections presented cellular necrosis, vacuolations and cellular infiltrates (Fig. 11). Group C, 8 weeks sections presented distortions in parenchymal tissue architecture, severe and generalized cellular necrosis with vacuolations (Fig. 12). Also, group D, 2 weeks sections presented distortions in parenchymal tissue architecture, edematous changes, inflammatory and necrotic cells with vacuolations, hemorrhagic exudations and eosinophilic cells-indicative of cellular degeneration (Fig. 13, 14). Group D, 4 weeks sections presented distortions in parenchymal tissue architecture with hemorrhagic exudations, degenerative fatty changes and inflammatory and necrotic cells with vacuolations (Fig. 15).

Fig. 7(a-d):

Test group B (8 weeks) sections (Liver, H&E×400) showing degenerative fatty changes (triangular arrow) (a) with cellular necrosis (encircled) (a and b), inflammatory cell infiltration (squared) (c) and vacuolations (b, c and d)

Fig. 8(a-c):

Test group C (2 weeks) sections (Liver, H&E×400) showing cellular necrosis and vacuolations (squared) (a, b and c) and hemorrhagic exudations (encircled) (b)

Group D, 6 weeks section presented distortions in parenchymal tissue architecture, necrotic cells with vacuolations (Fig. 16). Group D, 8 weeks sections presented distortions in parenchymal tissue architecture and severe cellular necrosis with vacuolations (Fig. 17). The severity pattern observed in the test groups indicates that the changes in the tissue architecture of the liver were dosage and duration dependent.

DISCUSSION

Many believed that smokeless tobacco is a safe substitute for smoking, however, the result of this study indicates the contrary. At 2 weeks early stage of the study, edematous changes, severe parenchymal erosion, cellular necrosis, vacuolations, hemorrhagic exudations and severe hemorrhage, distortions in parenchymal tissue architecture, inflammatory and necrotic cells with vacuolations and eosinophilic cells-indicative of cellular degeneration were observed in group B, C and D which shows hepatic alterations Fig. 2, 3, 8 and 9.

Fig. 9(a-d):

Test group C (2 weeks) sections (Liver, H&E×400) showing cellular necrosis, vacuolations and severe haemorrhage

Fig. 10(a-d):

Test group C (4 weeks) sections (Liver, H&E×400) showing cellular necrosis, vacuolations and severe cellular infiltration with haemorrhage

These hepatic changes are usually seen in hepatitis (chronic active hepatitis)²³, alcoholic hepatitis, liver cancer, cigarette smoking, drug and herbal abuse and other liver diseases and these indicate potential tobacco snuff-induced hepatitis. Smokeless tobacco-induced inflammation of the liver hepatocytes and causes cellular degeneration and blockage of liver sinusoids^1,18,24. Interestingly, Ugbor et al.⁶ reported tobacco snuff-induced severe acute and chronic hepatic profile alterations that are dosage and duration-dependent.

Fig. 11(a-f):

Test group C (6 weeks) sections (Liver, H&E×400) showing cellular necrosis (encircled), vacuolations (triangular arrows) and cellular infiltrates

Fig. 12(a-f):

Test group C (8 weeks) sections (Liver, H&E×400) showing distortions in parenchymal tissue architecture Note the severe and generalized cellular necrosis with vacuolations

Considering the subtle membrane changes that are usually induced by smokeless tobacco due to its ability to generate free radicals which have been implicated with hepatic injuries and which when persistent may cause liver cirrhosis, at 4 weeks stage of the study, groups B, C and D showed cellular necrosis, inflammatory cell infiltration, hemorrhagic exudations, degenerative fatty changes with vacuolations, severe cellular infiltration with hemorrhage and distortions in parenchymal tissue architecture with hemorrhagic exudations Fig. 5, 6, 10 and 15. These features agreed with the report of the Weissberg et al.²³ which stated that chronic active hepatitis reveals scar tissue and inflammatory cells, which disrupt the normal hepatic structure. In symptomatic cases, liver function tests are usually markedly abnormal, with evidence of hepatocellular injury (high transaminases) and diminished synthetic function (low albumin, prolonged prothrombin time), hence the report of Ugbor et al.^6,13 that tobacco snuff induced abnormally high liver function tests and inhibits the synthetic functions of the liver.

Illustratively, the scar tissue and inflammatory cells, which disrupts normal hepatic structure in chronic active hepatitis followed by abnormally high liver function tests indicate tobacco snuff potentials in causing tobacco-like hepatitis. Also, the degenerative fatty changes observed at this stage of the study indicate tobacco snuff-induced macrovesicular steatosis, a condition that describes abnormal retention of lipids within a cell. It also reflects an impairment of the normal processes of synthesis and elimination of triglyceride fat that is usually seen in cell toxins²⁵.

Fig. 13(a-f):

Test group D (2 weeks) sections (Liver, H&E×400) showing distortions in parenchymal tissue architecture

Fig. 14(a-c):

Test group D (2 weeks) sections (Liver, H&E×400) showing distortions in parenchymal tissue architecture with edematous changes (triangular arrows) Note the inflammatory and necrotic cells with vacuolations, as well as exudations as encircled (c)

As for the 6th weeks stage of the study, groups B, C and D presented edematous changes with hemorrhagic signs, cellular necrosis with vacuolations, cellular infiltrates and distortions in parenchymal tissue architecture Fig. 6, 11 and 16. These pathologic changes are symptoms of hepatitis^26,27 and other hepatic diseases. The severity of these changes showed that tobacco snuff is capable of inducing dosage and duration-dependent progressive hepatic damage through a mechanism that may be associated with eosinophilic cell and inflammatory cell infiltration into the liver, which then may trigger hepatic fibrosis.

Finally, at 8 weeks the result showed degenerative fatty changes with cellular necrosis, inflammatory cell infiltration and vacuolations and distortions in parenchymal tissue architecture with severe and generalized cellular necrosis throughout the test groups B, C and D Fig. 7, 12 and 17. Imperatively, Ugbor et al.⁶ reported the severity of tobacco snuff on hepatocytes due to the observed elevation of GGT in the previous study. These high levels of GGT were seen in patients with primary or secondary liver cancer and Rosalki et al.²⁸ also reported that liver necrosis and liver tumor are risk factors for elevated GGT.

Fig. 15(a-f):

Test group D (4 weeks) sections (Liver, H&E×400) showing distortions in parenchymal tissue architecture with hemorrhagic exudations (e) and degenerative fatty changes (triangular arrow, f)

Fig. 16(a-f):

Test group D (6 weeks) sections (Liver, H&E×400) showing distortions in parenchymal tissue architecture Note the necrotic cells with vacuolations

More and more, the Salunke et al.²⁹ revealed smokeless tobacco-induced cirrhosis of the liver and goblet cell hyperplasia of the small intestine.

Confirmative, the severe and generalized cellular necrosis observed implicates possible tobacco snuff-induced liver tumor. Meanwhile, severe cellular infiltration, vacuolations and distortions in parenchymal tissue architecture portray progressive hepatic destruction that is dosage and duration-dependent. It is worthy of note that tobacco snuff-induced hepatic damage is without doubt caused by its ability to generate free radicals, constitutive carcinogenic components, harmful heavy metals and 23 polycyclic aromatic hydrocarbons that are implicated in increased activity of serum hepatic parameters^6,7,30 and the progression of macrovesicular steatosis showed retrogression in the liver’s metabolic functions. The alterations observed in this study indicate that tobacco snuff is harmful and not good for human consumption.

Fig. 17(a-d):

Test group D (8 weeks) sections (Liver, H&E×400) showing distortions in parenchymal tissue architecture Note the severe cellular necrosis with vacuolations

CONCLUSION

Tobacco snuff causes progressive hepatic destruction with associated systemic distortions. In addition, the pattern of hepatic destruction is dosage and duration dependent with severe retrogression in liver metabolic functions.

SIGNIFICANCE STATEMENT

Tobacco snuff is consequentially unsafe for health and it also showed the destructive nature of tobacco snuff and the new health condition ‘Tobacco Snuff Hepatitis’. This study reveals the possible cause of most unknown hepatic disorders with devastating health consequences.

ACKNOWLEDGMENTS

We appreciate the entire staff of Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), Ile-Ife, Osun State, Nigeria for their support in allowing us to make use of their laboratory. The authors acknowledge the assistance provided by the staff of Anthonio Research Center, Ekpoma, Edo State, Nigeria, for their unconditional help during the experimental stage of this study.