||Thesis Name: Chemical and Toxicological Properties of Afzal, The Common Smokeless Tobacco Product In Oman
Researcher: Nawal Al-Azi Al-Mukhaini
Adviser: Prof. Taher A. Ba-Omar
Afzal is the common name for the illegal and discreetly sold smokeless tobacco product (STP) in Oman. It is commonly used by youth and teenagers. It is believed to contain many carcinogens and toxic constituents to body organs. The aims of this study are to analyze chemically a random selected Afzal sample for its harmful ingredients in accordance to the international routinely checked points in STPs and to assess the toxicity effects in the susceptible rat tissues esophagus, liver, kidney, testis & ovary treated with different doses of Afzal in adult and young groups of both genders. The chemical analyses of Afzal include the following analytes; pH level, moisture content, total and free nicotine, the carcinogenic Benzo[a]Pyrene (BaP), seven anions [nitrite (NO2-), nitrate (NO3-), Chloride (Cl-), Fluoride (F-), Bromide (Br-), Phosphate (PO4-3) and Sulphate (SO4-2)], five heavy metals [Cadmium (Cd), Chromium (Cr), Lead (Pb), Arsenic (As) and Nickel (Ni)] and four types of the carcinogenic Tobacco-Specific Nitrosamines (TSNAs) [4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB)]. Those analytes were tested using the following methodologies; Federal Register of CDC-USA (for the first two analytes), Gas Chromatography-Mass Spectrometry (GC-MS) (for the subsequent two analytes), Ion Chromatography (IC), Inductively-Coupled plasma-Mass Spectrometry (ICP-MS) and High performance Liquid Chromatography-tandem mass spectrometry (HPLC/MS-MS) respectively. While, the toxicological investigations of the same sample of Afzal were performed through two treatments (4 weeks and 8 weeks) for 144 Wistar albino rats, (n =72) young (4 weeks old) and (n=72) adult (20 weeks old) of both genders weighing between 60-80 g and 150-240 g respectively. The animals received aqueous extract of Afzal orally mixed in their drinking water and prepared freshly every week according to lethal dose of nicotine (LD50). They were divided into three main groups; low dose (3 mg Nicotine/kg body weight/day), high dose (6 mg Nicotine/kg body weight/day) and control (receiving distilled water instead of Afzal extract). At the treatment termination the animals were euthanized and their blood was collected for biochemical investigations and the following organs; esophagus, liver, kidney, testis and ovary were dissected out for histopathological investigations. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed for the liver function; blood urea nitrogen (BUN) and creatinine (CRT) were assayed for the kidney function while levels of testosterone and estradiol were assayed for fertility impairment.
The results showed Afzal has pH of (10.46), moisture content (52%), nicotine (48.77 mg/g total and 48.59 mg/g free), NO3- (8.79 mg/g), Cl- (33.17 mg/g) and Cr (15.75 µg/g). In addition, Afzal contained the two dangerous forms of TSNAs (NNN=1.205 and NNK=1.015 μg/g) and their summation exceeding the regulatory limits proposed by World Health Organization (WHO) (2 µg/g). The other analytes appeared in trace amounts but, BaP was not detected. While the toxicological investigations revealed reduced liver and kidney functions expressed as a significant increase in the serum’s ALT, AST, BUN and CRT levels (P<0.05) in both Afzal-treated groups (low and high doses) compared with the control. Moreover, Afzal causes fertility impairment manifested by a significant decrease in the testosterone and estradiol levels of both Afzal-treated groups compared with the control. The histopathological findings support and confirm the biochemical results and revealed the initial and serious degenerative alterations of periportal fibrosis in liver and edematous and calcified changes in renal glomerulus among Afzal-treated groups. The main reproductive damaging effects were reduction in the population with deformed organization of the germ cells and fatty and fibrous degenerations in testis and ovary. In addition, Afzal caused weight loss in the treated animals compared with the control. However, there was no structural damage of the esophagus seen in Afzal treated rats. Evidently, the chemical analyses reflect the fact that Afzal’s users may develop addictiveness and organ carcinogenicity due to high nicotine content and carcinogens. Toxicologically, Afzal was found to cause potential noxious effects appeared as dose-dependent functional and structural damage to the biochemical and histological profiles of liver, kidney, testis and ovary. Therefore, urgent national regulations and tough legislations of the illegal selling of Afzal along with health education and awareness of Afzal and its health risks are highly needed.