Antioxidant Trace Metals among Roadside Petrol Dispensers in Gombe State, Nigeria

Background: The number of people found to be exposed to gasoline is of public health concern. In developed countries, unlike developing countries, measures are taken to dispense gasoline quickly and safely to reduce exposure. In Nigeria, roadside dispensers are a common sight, often with no any form of protection. This leads to high levels of exposure with the consequences including oxidative injuries. As cofactors in antioxidant reactions, antioxidant trace metals are consumed in the process of converting the free radicals generated by petrol. Methods: Antioxidant trace metals (zinc, manganese, copper, and selenium) were compared between 90 road side dispensers of gasoline and 90 matched controls. Plasma zinc, selenium, manganese and copper ware analyzed using Atomic Absorption Spectrophotometer (AAS). Original Research Article Adamu et al.; BJMMR, 14(3): 1-7, 2016; Article no.BJMMR.24251 2 Results: The mean age of the exposed and control groups are 29.03±3.7 and 29.24±3.5 years respectively. Antioxidant trace metals zinc (64.7±6.09 μg/dl), manganese (7.93±1.32 μg/dl) and copper (77.8±7.38 μg/dl) of the exposed were significantly (p< 0.001) lower than the controls (100.2±9.58 μg/dl, 9.22±1.19 μg/dl and 103.8±20.09 μg/dl respectively). Selenium (0.03±0.008 μg/dl) was significantly (p< 0.001) higher in the exposed group compared to the controls (0.01±0.0023 μg/dl). Conclusion: This study has demonstrated lower level of antioxidant trace metals in roadside dispensers of gasoline compared to the controls. This indicates that those exposed are probably at greater risk of developing chronic diseases associated with increase oxidative stress. Enforcing legislation on roadside gasoline dispensing may reduce the risk.


INTRODUCTION
Gasoline as one of the most frequently utilized chemicals. It contributes much of the occupational exposure to toxic chemicals which is a major public health concern worldwide [1], the frequency is increasing due to rapid development in technology [2]. The risk of exposure is even greater in the developing countries, compared to the developed countries. Limited facilities for safe handling of such toxic substances and lack of knowledge of reducing the over exposure and the toxic effects of such chemicals contribute to the increase in the exposure in the developing nations. Poverty has also made people to engage in such occupations without consideration to the health effects of such occupations. Roadside gasoline dispensing is a common occupation in Nigeria most especially in the Northern parts. Lack of gasoline pumps and other protective means may lead to high levels of exposure to gasoline vapors among the roadside gasoline dispensers leading to production of ROS that may consume antioxidants including antioxidant enzymes and their cofactors like antioxidant trace metals.
It is established that gasoline is a very volatile substance, with many organic and inorganic constituents, [1] which when activated, lead to continuous production of reactive oxygen species (ROS) and consumption of antioxidants in the body. This leads to injury and diseases by causing damage to DNA, RNA, and proteins by chemical reactions such as oxidation, nitration, and halogenation leading to genetic modification and alteration in the functions of important lipids, enzymes and other proteins [3][4].
To prevent this injury, the body has developed a robust mechanism using substances known as antioxidants. The antioxidant can either be an enzyme like catalase, superoxide dismutase, glutathione peroxidase and thioredoxin reductase or a vitamin like beta-carotene, vitamin C and vitamin E. The antioxidant enzymes utilize antioxidant trace metals (selenium, zinc, copper, manganese) as cofactors in reactions leading to detoxification of the ROS in which both the enzymes and their cofactors including antioxidant vitamins and trace metals are consumed [5][6][7]. The expected increase turnover of the antioxidant enzymes as a response to the toxic ROS generated by exposure to gasoline may lead to the consumption and low levels of the antioxidant trace metals.
In Nigeria, there is paucity of literature on the antioxidant levels among roadside dispensers of gasoline. Therefore, understanding and having knowledge of the levels of antioxidant trace metals among these people will help in generating data to be used in the prevention and timely intervention of chronic gasoline toxicity especially in roadside dispensers of gasoline.
We therefore hypothesized that long-term occupational exposure to gasoline is associated with decreased antioxidant trace metals. The aim of the study, therefore, was to determine and compare the plasma levels of antioxidant trace metals (selenium, zinc, copper, manganese) between roadside dispensers of gasoline and controls in Gombe state, Nigeria.

Setting
This study is conducted in the department of Chemical Pathology, University College Hospital, Ibadan. The hospital is the premier teaching hospital in Nigeria and serves as a major referral center in Nigeria. The subjects in the study are roadside petrol dispensers in Gombe State, North-East Nigeria. The study was conducted in 2012.

Design
This is a retrospective cohort study approved by the joint Ethical Review Committee of the University of Ibadan/University College Hospital, Ibadan, recruiting otherwise healthy known roadside dispensers of gasoline in Gombe state using multi-staged sampling method. Age and sex matched controls were consecutively recruited from the same environment.

Inclusion Criteria
Only apparently healthy, full time roadside dispensers of gasoline that are one year an above in the trade were included in the study.

Exclusion Criteria
People who have other conditions that can cause oxidative stress were excluded. These include those working in painting, welding, battery, auto mechanics industries [8] and petrol station attendants were also excluded from the study. Patients who have other acute or chronic illness such as diabetes, chronic renal failure and hypertension were excluded from the study. Cigarette smokers and patients taking multivitamins supplements and antiretroviral treatment were also excluded from the study [9-10].

Selection of Controls
Age and sex matched controls were selected from the general population in the same environment.

Anthropometric Measurements
Height: -This was measured to the nearest centimetre against a flat, vertical surface with the subjects standing upright. A sliding headpiece was brought to the vertex of the subject's head and the reading at this level was taken. Their waist circumferences to the nearest 0.5 cm, Hip circumferences, waist to hip ratio and blood pressure were measured using standard procedures. Random plasma glucose was done using a glucometer. The questionnaires were administered to participants who were asked to fast for sample collection the next morning.

Sample Collection and Laboratory Procedures
5mls of fasting venous blood was collected from each of the 180 participants into a heparinised plastic tube. Plasma was separated by centrifugation frozen within an hour of collection till the time of analysis.

Statistical Analysis
The data was analyzed using SPSS version 20.00. Qualitative data were reported using percentages. The mean, standard deviation, skewness and kurtosis were used to measure the normality of distribution of the quantitative variables. The mean (SD) was reported for quantitative data and comparison was made between the cases and controls.
The normally distributed variables were compared between the two groups using student T-test. The level of significance was fixed at the 5% probability level. Pearson correlation coefficient was used to establish correlation between antioxidant vitamins and the duration of exposure among the exposed groups.  Table 2 shows the duration of exposure among the exposed population. All the continuous variables were normally distributed and therefore parametric analysis was used.  Table 3 shows equality of means in terms of age, blood pressure and other anthropometric characteristics between the cases and controls. This is to remove their confounding effects on the outcome of the study.

DISCUSSION
The objective of this study was to evaluate the plasma levels of antioxidant trace metals among roadside dispensers of gasoline. Volatile constituents of gasoline, when inhaled, will lead to continuous production of reactive oxygen species (ROS) and consumption of antioxidants. Since oxidative stress is an imbalance between the rate of production of ROS and their removal by antioxidants, oxidative stress can be assessed by assessing either the increase of production of ROS and its oxidizing effects on proteins, lipids and nucleic acids or by decreased levels of antioxidants [12][13]. This study therefore, compared the plasma levels of antioxidants trace metals between the roadside dispensers of gasoline and controls and also to determine the effect of the exposure period on the antioxidants.
High doses of petrol fumes are expected among roadside dispensers of petrol because they are less protected than petrol station attendants. This is because they use mouth to create a vacuum pressure to dispense the product through pipes into the receivers instead of pumps. They also, almost always, stay by the road side waiting for their customers which in itself is associated with increased levels of exposure to gasoline and its constituents [14].  Remarkably, with the exception of selenium, this study found a significantly lower level of antioxidant trace metals in the roadside gasoline dispensers than the control groups. This may be because as cofactors of the antioxidant enzymes, manganese, zinc and copper, are continuously utilized to produce the antioxidant enzymes that participate in the detoxification of the ROS [15][16].
Furthermore, previous studies have also reported significantly lower levels of antioxidant antioxidant enzymes, superoxide dismutase and glutathione peroxidase, and their cofactors including antioxidant vitamins in gasoline exposed subjects than in controls [5,17]. Zinc, manganese and copper, as components of SOD are closely inter-related to antioxidant functions, It is likely, therefore, that their deficiency may lead to impaired free-radical scavenging mechanisms thereby increasing oxidative stress in the exposed group [3,18]. This may explain the increase in the risk of chronic diseases found in this group when compared with controls [19].
The finding in this study, of significantly higher level of selenium among the exposed than the controls is not consistent with what is expected. However, some studies have found higher levels of some antioxidant trace metals in association with low antioxidant enzymes in variable combinations. For example, a study found higher levels of selenium with lower levels of total antioxidant capacity (TAC) among cases (epileptics) compared to controls [20] Similar pattern was observed in a study where selenium was higher despite lower levels in other antioxidants in patients with vitiligo compared with controls in which the significance of higher selenium level was described as unknown [21][22]. Similar findings were made in many studies [23][24][25][26]. Assaying antioxidant enzymes would have given an opportunity to compare the plasma levels of the selenium dependent enzyme glutathione peroxidase (GPx) and the selenium levels in the plasma. This would have predict the possibility of a switch from selenium dependent glutathione peroxidase in the exposed group to the selenium independent isoform which could have accounted for the higher levels of selenium in them [22]. But this is unlikely considering the young age nature of the study participants since the glutathione peroxidase switch is age dependent [22]. Another possibility is by sulphur in the gasoline fumes substituting selenium from selenocysteine which does not happen among the controls [27].
The negative correlation between antioxidant trace metals and the duration of exposure (not statistically significant) further demonstrated the possibility of gasoline mediated decrease in antioxidant trace metals and possibility of increase oxidative stress in the roadside dispensers of gasoline.
There is, therefore, a strong need for going further to find out the effect of antioxidant supplementation on the makers of oxidative stress in the non-gasoline station dispensers of gasoline. This is because many studies have shown a decrease in oxidative stress makers in people with oxidative stress after antioxidants supplementation [28]. If found to be useful; antioxidant supplementation can be advocated to reduce the risk of oxidative stress among the people that are exposed to gasoline.

CONCLUSION AND SUMMARY
This study has demonstrated a strong relationship between exposure to gasoline and decrease in antioxidant trace metals. This study may be the first of its kind among roadside gasoline dispensers. There is, therefore, a strong need to do a cohort study that involves following the exposed individuals to monitor progression of the oxidative stress makers or their reduction by antioxidant supplements. From the study, none of the exposed people use any form of protection, indicating limited knowledge of the risk involved in their occupation. There is, therefore, a great need for public awareness on the risk involved in road side gasoline dispensing and the ways of minimizing it.

CONSENT
The study was explained to the participants in the language they understand and written consent was obtained from them (or other approved parties) for publication of this paper and accompanying images.