POLYCYCLIC AROMATIC HYDROCARBON , HAEMATOLOGICAL AND OXIDATIVE STRESS LEVELS IN COMMERCIAL PHOTOCOPIER OPERATORS IN LAGOS , NIGERIA

Background: Human exposure to hazardous substances in the environment has been known to play an important role in the pathogenesis of some diseases. Photocopying machines have become a cheap source of self-employment in Nigeria. For obvious reasons the highest level of patronage is encountered in the campuses of educational institutions. However, the persons who operate the machines are always exposed to possible hazards associated with the job without protective devices. Objective: This study investigated the levels of oxidative stress, polycyclic aromatic hydrocarbon (PAH) and haematological parameters in blood samples of photocopier operators. Methods: The experimental procedure involved 50 consented subjects selected based on some criteria. The haematological parameters, oxidative stress and PAH levels were determined using standard methods. Results: The results showed no significant difference (p ≥ 0.05) in the haematological parameters between the test subjects and the controls. However, there were duration on the job (yrs) dependent significant decrease in the level of superoxide dismutase (SOD) of the photocopier operators compared with the controls (> 5 years p≤ 0.0001; 4-5 years p≤0.001). The level of reduced glutathione (GSH) was significantly decreased across all lengths of duration on the job compared with the controls. Conclusion: The findings in this study revealed increased level of oxidative stress in photocopier operators with no significant change in haematological parameters. The health implication of operating photocopiers call for quick health education and intervention tailored to monitoring and guiding the photocopier operators. This will help to prevent or manage continuous exposure to the hazards of photocopying machines.


INTRODUCTION
Exposure of humans to hazardous substances in the environment has been known to play an important role in the pathogenesis of some diseases. 13][4][5] In addition, workers may also be exposed to additional toxic substances due to their social habits such as smoking, tobacco chewing and alcohol consumption.
As a result of massive unemployment and economic downturn, photocopying machines have become a cheap source of self-employment in Nigeria and for obvious reason the highest level of patronage is encountered in educational institutions. 6Most of the photocopy shops/centres in Nigerian universities exhaust one thousand sheets of paper daily for photocopying activities and minimum of 349,000 sheets of duplicating paper per day goes for photocopying in eight selected universities in South West Nigeria. 6e report of Gadhia et al 7 has also shown that photocopiers are used in many work places as a good source of employment and are common machines in Indian markets.The persons who operate the machines are always exposed to possible hazards associated with it.Toner which is the main component of the photocopying machine consists of carbon black (17%), polycyclic aromatic hydrocarbons (PAH) 8 , styrene, magnetite, nitropyrenes, benzene, toluene, other volatile substances and low melt polymer resins mixed with minute steel, silica or ferrite bead. 9he operators are exposed to toner (while reloading and unloading the machines) and to toxic gases like ozone, nitrogen dioxide, volatile organic compounds and extremely low frequency electromagnetic fields during their operation [8][9][10][11][12] .7] However, significant association was found between years of exposure, smoking, age, gender, alcohol consumption and high level of DNA damage. 17Reduced level of anti-oxidants like vitamin C and E and superoxide dismustase (SOD) and high activity of lipid peroxidation were detected in the blood samples of photocopier operators. 5The earlier study of Zhou et al. 5 suggested that ozone, which is one of the gasses released from photocopier toner, causes oxidative damage in copier operators.
Occupational autoimmune disease in photocopying machine workers has also been reported. 18aematological study of total red blood cells, total white blood cells, haemoglobin percentage and platelet count from photocopying machine operators did not reveal any untoward variations from normal values and measurement of their blood pressure showed normal pattern. 7e aim of the present study is to determine the level of oxidative stress markers in the blood of workers occupationally exposed to photocopying machines, evaluate the effect on blood cells, and assess the types and levels of polycyclic aromatic hydrocarbon (PAH) in their plasma.To the best of our knowledge, similar or related studies in this category have not been carried out among Nigerian photocopier operators.It is hoped that the findings will lead to appropriate recommendations that will reduce the long term health implications of operating photocopying machines.

Study Area
The two main study areas are the University of Lagos Main Campus, Akoka and the University of Lagos, College of Medicine, Idi-Araba, Lagos, Nigeria.University of Lagos is located in South West Nigeria bordered by the panoramic Lagos lagoon.The total students' population is about fifteen thousand with an estimated two hundred and seventy commercial photocopier operators working in both campuses of the University.

Study Population
The experimental procedure involved 50 consented subjects.The selection criteria of the study subjects were based on the outcome of the preliminary survey carried out.The photocopier operators voluntarily signed an informed consent form at the beginning of the survey and the consented operators responded to an elaborate questionnaire to determine their sociodemographical data and some job related issues.One hundred and twenty-six questionnaires were returned out of the two hundred and sixty-one that were distributed.Some categories of respondents were excluded from the experimental procedure due to their work experience in factories that are noted for harmful emissions similar to those of photocopiers, females for physiological reasons, smokers and alcoholics because of their increased oxidative stress tendency.Also the subjects selected did not include those that had been taking any anti-oxidant on a regular basis or had been exposed to any kind of radiation for 12 months before the exercise.

EXPERIMENTAL PROCEDURE Study Group
The fifty selected subjects were divided into five categories.The first four categories consist of 10 subjects each of photocopier operators.The duration of the subjects on this job formed the basis of their grouping into different categories (2-3 yrs, 3-4 yrs, 4-5 yrs and >5 yrs).The control subjects also consist of 10 consented students of the University who were neither smokers nor alcoholics.

Sample Collection
Blood samples were collected by skilled hands under sterile conditions through venipuncture.About 3mls of blood were transferred into sodium heparinised bottles for centrifugation and the plasma obtained stored at -30 o C for antioxidants and lipid peroxidation assays.Another 2mls were transferred into EDTA bottles for haematological analysis.While 1ml of the blood samples was withdrawn into the plain bottles for polycyclic aromatic hydrocarbon (PAH) assay.

GC Analysis
The standard procedure was used to extract the polycyclic aromatic hydrocarbons from the biological sample.The 7890A, MS5975C models of GC-MS system and 7683B with 8 auto-samplers injector supplied by Agilent Technologies (USA) were used for GC analysis.The column used was HP-5 MS with a length of 30 metres, internal diameter of 0.32 mm and film of 0.25µm fused silica capillary column and helium as the carrier gas.The pressure flow was 10 psi to give approximate flow rate of 1ml/min.The injector and transferred line were maintained at 290 o C and 250 o C respectively.All injection volumes were 1µl in a split mode.The column initial temperature was held at 100 o C for 4 mins rampled to 300 o C at a rate of 8 o C/min, and held at 300 o C for 10 mins.The mass spectrometer was used in electron ionization mode and all spectra were acquired using a mass range of 50-400 m/z and an automatic gain control.

Determination of Air Emission/Concentration
The determination of air emission/concentration at photocopier operation site was carried out by using Testo 350 emission analyzer.This instrument was obtained from the Lagos State Environmental Protection Agency, Ikeja, Lagos, Nigeria.The emission testing relies on proper hardware coupled with accurate measurements.The sensors were integrated, flow rate controlled and exhaust gas conditioned for appropriate detection of air emission gases.The operation was carried out by clicking the application icon and the air emission analyzer automatically began processing the levels of air emission gasses at the site of operation.

Statistical analysis
The socio-demographical data were presented in frequency distribution tables with percentages and experimental results were presented as mean ± S.E.M. Statistical significance between the control group and the test groups were analyzed by means of Student's ttest.P values less than 0.05 were considered significant.

RESULTS
Table 1 results show the socio-demographic data of the photocopier operators.The highest age distribution (60.30%) of the subjects was found to be between 15-25 years and the lowest (1.6 %) was found to be between 46-55 years.There were more male subjects (61.10%) than female and most of them had secondary education (67.50 %).
The results further showed that 31.70% of the subjects had spent less than 1 year on the job as photocopier operators while 20.60 % had spent more than 5 years on the job.Slightly larger proportions of the subjects (52.40 %) like their job as photocopier operators (Table 2).There were duration on the job (yrs) dependent significant decrease (> 5 yrs p ≤ 0.0001; 4-5 yrs p ≤ 0.001) in the level of SOD of the photocopier operators compared with the control (Table 4).The level of GSH significantly (p ≤ 0.05) decreased across all length of duration on the job (years) compared with the control.However, only the subjects who had stayed for more than five years on the job as photocopier operators had significant (p ≤ 0.05) decrease in the level of CAT (348.80 ± 78.96 U/mg) compared with the control (595.00 ± 30.43 U/mg).There was also duration on the job dependent significant increase (3-4 yrs p ≤ 0.05; 4-5 yrs p ≤ 0.001; >5 yrs p ≤ 0.0001) in the level of lipid peroxidation (MDA) of the photocopier operators compared with the control (Table 4) The common health problems of the photocopier operators as shown in Figure 1 are eye irritation, headache, dizziness and catarrh.The haematological parameters results (Table 3) showed no significant difference (p ≥ 0.05) in the levels of haemoglobin, white blood cells, neutrophils, lymphocytes, monocytes and eosinophils between the photocopier operators and the control subjects.The combination of retention time match and mass spectral match of GC/MS of the plasma of photocopier operators and control subjects did not show any polycyclic aromatic hydrocarbon (Figure 2).
Analysis of the air emission showed no detection of CO, NO 3 , NO, NO 2 , CO 2 , SO 2 , and hydrocarbons at the sites using electric power while the air emission at photocopier operation site using petrol generator showed significant high level of hydrocarbons (152 ppm) in comparison with the environmental protection agency level (0.008 ppm).

DISCUSSION
Photocopiers have been a good source of job opportunity for most secondary school leavers in Nigeria as it is easy to operate and commercially needed in almost all our tertiary institutions.This same phenomenon occurs in India where photocopiers are good source of employment and most common machines in Indian markets. 7The people who operate the machines are exposed to possible hazards associated with it.
'Toner' is the main component of photocopying machine that mainly consist of carbon black (17%), polycyclic aromatic hydrocarbons (PAHs), styrene, magnetite, nitropyrenes, benzene, toluene, other volatile substances and low melt polymer resins mixed with minute steel, silica or ferrite beads.Besides these, ozone, nitrogen dioxide, volatile organic compounds like 1-1, biphenyl p-dichlorobenzene pyrolbenzene and tetrachloroethylene aldehydes are also released into the atmosphere by the machines while in operation. 7 is well known that superoxide dismutase, catalase and reduced glutathione are important endogenous antioxidants in human bodies.They play important roles in scavenging free radicals like superoxides (O .), hydroxyl group (OH) as well as reactive oxygen species like singlet oxygen ( 1 O 2 ) and hydrogen peroxides (H 2 O 2 ) which are excessively generated in human bodies either naturally or upon exposure to environmental or occupational hazardous substances. 23- 27; 28-33 The results obtained from this study had shown that there is a significantly higher level of oxidative stress in workers occupationally exposed to photocopiers compared to the control subjects.This finding agrees with earlier findings of Zhou et al. 5 who demonstrated that photocopying machine workers suffer an increased level of lipoperoxidase in plasma and red blood cells as well as reduced levels of superoxide dismutase, catalase and glutathione peroxidase.This may be correlated to work load as expressed by the number of machine activations.
This present study revealed duration of occupation dependent decrease in the level of SOD of the photocopier operators compared with the control and also significant increase (3-4 yrs p ≤ 0.05; 4-5 yrs p ≤ 0.001; >5 yrs p ≤ 0.0001) in the level of lipid peroxidation (MDA) of the photocopier operators compared with the control.These observations may be due to the presence of ozone, PAH and ultraviolet emission from the photocopying machines which may induce reactive oxygen species in exposed tissue.This leads to increasing production of highly toxic hydroxyl radicals and increasing chronic oxidative stress in the bodies of the photocopier operators. 5The health implications of photocopiers had earlier been shown by Bar-Sela and Shoenfeld 18 ; Goud et al. 16 to include autoimmune disorder and DNA damage.This present study has also shown that eye irritation, headache, dizziness and catarrh were common health complaints of photocopier operators.
However, the haematological parameters of the photocopier operators were normal compared with the control subjects.This finding corroborates the work of Gadhia et al 7 that showed Indian photocopier operators to have normal haematological parameters and blood pressure values.
The non-detection of polycyclic aromatic hydrocarbons in the plasma of photocopier operators as shown in this study is unexpected and may be due to the method of analysis used, length of exposure or other confounding variables.Thus, further work may be done using more specific method of PAH analysis.It is painful to know that despite the awareness (46 %) of the potential hazards of photocopiers; most of the operators (66.7 %) were not using any protective device.This observation calls for a quick health education and intervention tailored to monitoring and guiding photocopier operators so as to avoid continuous exposure to the hazards of photocopying machines.

Figure 1 Figure 2
Figure 1 Common health problems of photocopier operators

Table 1
Socio-demographic data of photocopier operators

Table 2
Job related information about the photocopier operators

Table 3
Haematological parameters of photocopier operators

Table 4
Oxidative stress parameters of photocopier operators