Introduction

Oral cancer is the fifth most common cancer worldwide (Parkin et al., 1993) & is one of the most common malignancies in India accounting 30-40% of all cancers.⁽¹²⁾   Males, particularly over 40 years, are affected twice as often as women.  Free-radical induced damage is an important factor in oral carcinoma.  Antioxidants, particularly vitamin C, come to rescue exhibiting altered levels of oxidants & antioxidants during the pathogenesis of disease.

Vitamin C is also a highly effective non-enzymatic antioxidant.  Several possible mechanisms of action of ascorbic acid in cancer prevention have been described extensively elsewhere.⁽¹⁰⁾ It plays a major role in free-radical scavenging & protection against lipid peroxidation. It appears to have a role in sparing or reconstituting the active forms of vitamin E⁽³⁾ & spares other important antioxidants.⁽⁵⁾

Plasma levels of anti-oxidants are reliable indicators of the antioxidant status, because they reflect the bioavailability as well as increased utilization to counter lipid peroxidation. Furthermore anti-oxidants levels in plasma are also influenced by life style factors such as diet & tobacco.  Tobacco contains a high level of TSNA ⁽¹³⁾ increased formation of which might be expected in the oral cavity of pan & gutkha chewers with poor oral hygiene.  Chewing is known to cause local trauma & injury to oral mucosa due to its abrasive nature which may lead to injury‐related oxidative stress & subsequent ROS generation, which in turn during chronic exposure can lead to malignancy in oral cavity.

Whether chewing tobacco reduces vitamin levels is unknown.   There have been no previous reports on vitamin C levels in tobacco chewers. Theoretically, one might expect chewing to have less of an effect on antioxidant vitamin status than smoking does, because it exposes the tobacco user to smaller quantities of free radicals. On the other hand, chewers are exposed to greater quantities of precursors of carcinogenic nitrosamines, & additional vitamins C may be needed to help prevent nitrosation.

Materials and Methods

Eligible cases were 30 healthy subjects & 30 histopathologically-diagnosed cases (25 males & 5 females) of OSCC (tongue 2, buccal mucosa = 24, & hard palate = 4), particularly grade I (n = 19) & grade II (n = 11).  All of them were in either age group 25-50 years or 51 & above years.  Blood samples were collected from the O.P. Department of Surgery, Sasoon Hospital, B.J. Medical College, Pune, Maharashtra, India.  A detailed history of patients included age, sex, duration of disease, duration of chewing habits, socioeconomic background, etc.

Inclusion Criteria

Histopathologically-diagnosed & confirmed cases of squamous cell carcinoma of oral cavity [grade I & grade II].

Control cases were healthy, i.e. free from any disease/abnormality/or any habit including tobacco chewing.

All cases had a well-known history of tobacco-chewing habit [gutkha & BQ/pan].

Exclusion Criteria

Grade III & IV patients;

Those with carcinoma of lip, pharynx, & larynx, tonsils, minor salivary gland tumors, etc;

Those with any concomitant disorder such as diabetes mellitus, liver diseases, rheumatoid arthritis, etc or scorbutus;

Patients taking vitamins/antioxidants supplements.

Those having previous history of smoking or alcohol abuse.

Method:  Taking aseptic precautions, approximately 5 ml of blood samples were collected, from in appropriate sterile vials by venous arm puncture after overnight fasting & sera were separated by centrifugation at 3000 rpm for 10 minutes.  Estimation of serum vitamin C [Ascorbate] was carried out colorimetrically, within 24 hour of specimen collection, by the method of Ayekyaw (1978) where phosphotungstic acid first deprotienizes the sample & then react with ascorbic acid to produce blue color [660 nms].⁽¹⁾

Calculation:

Vitamin C [mgs/dL] =    O.D. [T] – O.D. [B] _{* Std Conc.}

O.D. [S] – O.D. [B]

Statistical Analysis:  Data were expressed as (mean ± SD). Statistical analysis [patient Vs control] was done using students’ t-test & the comparison between groups was performed with one way analysis of variance (ANOVA). P-value of less than 0.05 was considered as statistically significant.

Results and Discussion

Vitamin C levels showed a highly significant decrease in oral cancer patients as compared to control group which concluded an inverse relationship with the severity of disease (p < 0.001).  Similar results were obtained by Syed Sultan Beevi etal (2004)⁽¹⁵⁾  Low levels of vitamin C could be due to increased utilization of antioxidants to counteract free-radical mediated cell disturbances. This finding correlated with the findings of previous studies.^(13-16)

Low levels were noted in Grade II patients as compared to Grade I patients & the difference was statistically highly significant (p < 0.001) w r t control.  This may be due to shifting of ascorbic acid from blood to site of malignancy & thereby decreasing serum levels, possibly due to increased requirement in higher grades.  The development & progression of cancer evokes an increased requirement for ascorbic acid.  There is good evidence that high intake of ascorbic acid potentiates immune system in various ways & ascorbic acid may also offer some protection against variety of agents that cause cancer.  It is also believed that a strong immune system counteracts traps & eliminates cancer cells.  Thus ascorbic acid could exert this anticancer function indirectly.  It can stop the initiation or limit the cell proliferation or metastasis of various organs by a series of mechanism some of them at molecular level.  It is appropriate that it could be used as a therapeutic agent in cancer.  Use of ascorbic acid in therapy of cancer could be in the form of supportive or adjunct-therapy.  The determination of serum levels of ascorbic acid will indicate the status of oral cancer patients.

Levels were much lower in gutkha chewers than in pan chewers & the results obtained were also statistically highly significant when compared against control (p < 0.001).  These data indicate that tobacco chewers have increased demands for antioxidant nutrients & chewers may need to consume more vitamin C than nonusers do in order to meet their needs for these vitamins.⁽¹⁷⁾  Chewing might restrict the body for utilization of antioxidant nutrients like vitamins, as they should, which results in fall in levels of vitamin C.

All pan chewers developed low grade cancer ⁽¹¹⁾ while most gutkha chewers had intermediate grade.  Similarly in the present study, all pan chewers (n = 11) had low grade carcinoma.  Pan leaf is known to be rich in beta-carotene, which has the capacity to quench free radicals that are mutagenic ⁽⁷⁾ which probably delays the onset of oral carcinogenesis. Gutkha chewers developed more severe grade probably due to use of more dry weight of areca nut & tobacco as compared to other chewing products.  Mohommad Sami ⁽¹¹⁾, Babu et al ⁽²⁾ & Gupta & Ray ⁽⁸⁾ have described that gutkha chewers develop OSMF earlier & in a more severe condition than chewers, using other products.  It is possible that as areca nut has high alkaloid arecoline & tobacco ingredients like nitrosoamine, which are absorbed more in the patients, have more severity of the disorder.

Although dietary factors have been identified as having a possible association with oral cancer, accumulated scientific evidence that use of tobacco increases oral cancer risk far outweighs any evidence linking a deficient diet to increased risk.⁽¹⁴⁾  A low intake of vitamin C has been associated with an increased risk of cancers of the oral cavity. Patients who ingest high levels of vitamin C have half the risk of oral cancer as those with the lowest level of consumption.⁽⁶⁾

Table 1:  Mean ± S.D. Values:-

^aF cri 0.001 _{(2, 57)} = 7.96 (p < 0.001), **p < 0.001

Table 2:  Mean ± S.D. Values According to Chewing Habits :-

** p < 0.001, F cri 0.001 _{(2, 57)} = 7.96 (p < 0.001)

Conclusion

Low vitamin C levels significantly increases risk of oral malignancy. Also levels of vitamin C were found to be associated with tumor grades of patients, thus measurement of vitamin C in circulation of oral cancer patients may be helpful in assessing the tumor grades of oral cancer.  Tobacco, particularly, gutkha chewers have low levels of antioxidant vitamin C in serum.  Low antioxidant nutrient, particularly vitamin C, & tobacco can be regarded as major risk factors in progression of oral cancer. Future research should focus on analysis of risks in individuals in which one or more nutrients are low or high public health action should be directed towards increasing consumption of food containing naturally-occurring antioxidants.  Hence, larger sample size & further studies on this arena will elucidate more substantially, & will give, a new insight on vitamin C levels & oral carcinogenesis.

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