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Reversal of aminolevulinic acid dehydratase (ALAD) inhibition and reduction of erythrocyte protoporphyrin levels by Vitamin C in occupational lead exposure in Abeokuta, Nigeria
Environmental Toxicology and Pharmacology 20 (2005) 404–411
Reversal of aminolevulinic acid dehydratase (ALAD) inhibition and reduction of erythrocyte protoporphyrin levels by Vitamin C in occupational lead exposure in Abeokuta, Nigeria O. Ademuyiwa a,∗ , R.N. Ugbaja a , D.A. Ojo b , A.O. Owoigbe a , S.E. Adeokun a a b
Department of Biochemistry, University of Agriculture, Abeokuta, Nigeria Department of Microbiology, University of Agriculture, Abeokuta, Nigeria Received 12 December 2004; accepted 20 April 2005 Available online 27 June 2005
Abstract In order to investigate the toxic effects of lead during occupational exposure to this metal, the activity of aminolevulinic acid dehydratase (ALAD) and the concentrations of erythrocyte protoporphyrin (EPP) were determined in blood of various artisans in Abeokuta, Nigeria and these were related to blood lead levels. ALAD activity in the artisans was inhibited to varying extents. ALAD activity was inhibited to the tune of 77% in petrol station attendants while the lowest inhibition of 36% was obtained in the welders. EPP was also significantly increased in the artisans (p < 0.001). The highest EPP level of 241.57 ± 89.27 g/100 ml of red blood cell was observed in upholsterers. A significant (p < 0.001) negative correlation was observed between ALAD activity and blood lead levels on one hand (r = −0.631) and between ALAD activity and EPP on the other hand (r = −0.461). Administration of a daily dose of 500 mg ascorbic acid for 2 weeks reversed the lead-induced inhibition of ALAD. Increased EPP levels observed in the artisans also responded positively to the ascorbic acid supplementation. A significant reduction (p < 0.001) in blood lead was also observed in the artisans at the end of the 2-week ascorbic acid therapy. Our findings indicate that ascorbic acid may offer more advantages over the conventional agents for the treatment of lead poisoning, especially in cases where the subjects cannot be removed from the source of lead exposure. © 2005 Elsevier B.V. All rights reserved. Keywords: ALAD inhibition; Erythrocyte protoporphyrin; Artisans in Nigeria; Reversal; Ascorbic acid
1. Introduction Lead, the most ubiquitous toxic metal found in the earth’s crust, has been used by human beings for thousands of years and it is still one of the most widely used metals in industries (Pande and Flora, 2002). The health hazards of lead and its compounds have been the focus of scientific attention in many countries of the world (Ademuyiwa et al., 2002; Canfield et al., 2003; Lalor et al., 2001; Nriagu et al., 1997; Onunkwor et al., 2004). Numerous studies have demonstrated that lead is a common environmental and industrial pollutant that affects ∗
Corresponding author. Tel.: +234 803 357 5238. E-mail address: [email protected] (O. Ademuyiwa).
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virtually every system in the body (Ademuyiwa et al., 2002; Albalak et al., 2003; Canfield et al., 2003; Kentner et al., 1994; Onunkwor et al., 2004; Sakai, 2000). Neurobehavioral, hematologic, nephrotoxic and reproductive effects of lead have been observed in humans and other animals (Elinder et al., 1988; Goyer, 1991; Needleman et al., 1990; Rice, 1996). The immune system is not spared from the toxic effects of lead (Basaran and Undeger, 2000). One of the best known toxic effects of lead is its interference with heme biosynthesis (Alexander et al., 1998; Flora et al., 2003; Makino et al., 2000). Elevation of delta-aminolevulinic acid (ALA) level, due to inhibition of delta-aminolevulinic acid dehydratase (ALAD) enzyme, followed by elevation of protoporphyrins in erythrocytes and increased urinary excretion of ALA and coproporphyrin, are
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early effects associated with increasing lead concentration in soft tissues (Alexander et al., 1998; Flora et al., 2003; Sakai, 2000). Elevation of ALA results in overproduction of active oxygen species (Gurer et al., 1999; Neal et al., 1997). Because of its high sensitivity to lead, inhibition of ALAD has been used as an index of environmental lead pollution (Campagna et al., 1999; Chalevelakis et al., 1995; Flora et al., 2003; Tandon et al., 2001). Lead exposure studies (both environmental and occupational) is a well-trodden subject in developed countries (Canfield et al., 2003; EPA, 1986; Goyer, 1993; Meyer et al., 1998; Pirkle et al., 1994; P¨onka, 1998; Rice, 1996; Stromberg et al., 1995). In contrast however, such information is still relatively scarce in developing countries like Nigeria. In previous studies, elevated lead levels were observed in petrol station attendants and auto-mechanics in Abeokuta, Nigeria (Ademuyiwa et al., 2002; Dosumu et al., 2005; Onunkwor et al., 2004). Information about lead levels in other artisans in Nigeria is lacking. In addition, no study has been undertaken in Nigeria to determine the extent of inhibition of ALAD in those who are occupationally exposed to lead. For these reasons, ALAD activity and the concentration of erythrocyte protoporphyrin (EPP) were determined in blood of different groups of artisans in Abeokuta, Nigeria and these were related to blood lead levels. Petrol station attendants and auto-mechanics were included in the study in order to compare their ALAD levels with other artisans since they have been shown to be occupationally exposed to lead (Ademuyiwa et al., 2002; Dosumu et al., 2005; Onunkwor et al., 2004). The hypothesis was also tested whether ascorbic acid would remove lead-induced inhibition of ALAD.
2. Subjects and methods 2.1. Study population A total of 110 male subjects participated in the study. Control subjects were made up of staff and students of University of Agriculture, Abeokuta, Nigeria, while leadexposed subjects comprised of different artisans located in two mechanic workshops in the southern and northern parts of the city of Abeokuta, Nigeria. It is typical of mechanic workshops in Nigeria to find other groups of artisans located in these workshops in addition to the auto-mechanics, thereby complementing each other’s services. In these workshops, there is always a preponderance of auto-mechanics. After explaining the objectives of the study to them, a total of 92 artisans, including 50 auto-mechanics, consented to participate in the study. Seven male petrol station attendants from a petrol station in the southern part of the city were also included among the occupationally-exposed subjects. A careful history of their dietary habit and their job experience was taken. Table 1 summarises the study population.
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Table 1 Study population Subject
Number
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
11 50 8 2 2 7 15 7 4 2 2
All the subjects studied were non-smokers. Six of the auto-mechanics were alcoholics, taking one bottle of beer per day; five of the auto-mechanics, two panel beaters, two petrol station attendants and one auto-electrician, were occasional alcoholics, taking between one and two bottles of beer in 2 months. 2.2. Supplementation regimen The ascorbic acid (Mopson Pharmaceuticals, Lagos, Nigeria) was given as a daily dose of 500 mg ascorbic acid for 2 weeks to each subject. During ascorbic acid supplementation period, occasional visits were paid to the subjects to ensure compliance with the regimental protocols. 2.3. Sampling Blood samples were collected before and after ascorbic acid supplementation. Venous blood samples (about 10 ml) were collected from each subject by a trained medical laboratory staff into heparinised tubes. The blood samples were stored in a cooler box and then transferred to the laboratory for analysis. The two drivers were away on a journey on the day the second blood samples were to be collected; as such, no values were reported for them. 2.4. Lead analysis Lead was determined in whole blood by atomic absorption spectrometry (Buck Scientific AAS Model 210, Connecticut, USA). 1 ml of whole blood was digested with concentrated nitric acid and digests brought to a constant volume. External standardisation for lead was done by analysing a certified Spex Lead Standard (Spex Industries Inc., Edison, New Jersey, USA) along with the samples. Procedural accuracy of the lead analyses was also evaluated by spiking control samples with the Spex Lead Standard. In all cases, two determinations were made for each sample and the mean lead recovery rate of 98.37 ± 4.57% was obtained for all the analyses.
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2.5. Determination of ALAD activity
Table 2 Some demographic characteristics of the subjects
The activity of ALAD was also determined in whole blood less than 24 h after blood collection using the European Standardised Method described by Berlin and Schaller (1974). 0.2 ml of heparinised blood was mixed with 1.3 ml of distilled water and incubated for 10 min at 37 ◦ C. 1 ml of standard ALA was then added and the tubes were further incubated for 1 h. The reaction was stopped by adding 1 ml trichloroacetic acid (TCA) containing mercuric chloride (HgCl2 ). To 1 ml of the supernatant, 1 ml of Ehrlich’s reagent was added and the absorbance was read at 555 nm after 5 min.
Subject
Age in years
Job experience in years
Hours spent per day in workshop
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
26.00 ± 6.65 28.02 ± 10.72 32.00 ± 10.17 27.00 ± 7.07 39.50 ± 12.02 34.00 ± 19.56 29.53 ± 13.03 28.71 ± 3.99
– 11.64 13.25 7.50 4.00 15.71 12.47 3.57
8.64 11.62 11.25 11.50 10.00 11.14 11.20 14.29
29.25 ± 8.96 30.50 ± 0.71
12.38 ± 11.46 6.50 ± 2.12
11.25 ± 1.50 10.50 ± 2.12
37.50 ± 3.54
25.50 ± 0.71
11.50 ± 0.71
2.6. Determination of erythrocyte protoporphyrin (EPP) EPP was determined in whole blood using the method of Heller et al. (1971). Briefly, 2 ml of heparinised blood was mixed with 2 ml of acetone:ethylacetate mixture (1:9, v/v) in a 10 ml centrifuge tube. The mixture was then vortexed vigorously for about 1 min. Four millilitres of formic acid:diethylether mixture (1:9, v/v) was then added and the mixture vortexed again for another 1 min. This was then centrifuged at 5000 rpm for 5 min. The supernatant was decanted into a 10 ml graduated centrifuge tube and the precipitate reextracted with a second 4 ml formic acid:diethylether mixture. To the combined supernatants, 2 ml 1.5 M HCl was added and this was vortexed vigorously for 30 s. The volume of the (lower) HCl layer was recorded. The HCl layer was then transferred into a small test tube with a long fine tip pipet. The absorbance of this solution was then read at 380, 407 and 430 nm against 1.5 M HCl blank with a Jenway 6405 UV/Visible spectrophotometer (Jenway Ltd., Felsted, Dunmow, Essex, UK). 2.7. Statistical protocol Results are expressed as mean ± S.D. One-way analysis of variance (ANOVA) followed by the LSD (least significant difference) test were used to analyse the results with p < 0.05 (Sachs, 1983). The relationships between ALAD activities and lead exposure measurements on one hand; and between ALAD activities and EPP concentrations on the other hand, both before and after the 2-week ascorbic acid supplementation, were analysed using Pearson correlations (Sachs, 1983). The relationships between blood lead levels and EPP both before and after ascorbic acid supplementation were also analysed using Pearson correlations (Sachs, 1983).
3. Results Some demographic characteristics of the subjects are depicted in Table 2. Their ages ranged between 18 and 66 years. The years of experience on the job for each group shows a wide variation, ranging from 0.7 to 53 in the automechanics and 1.5 to 5 in the petrol station attendants. It
± ± ± ± ± ± ±
10.92 11.18 0.71 2.83 19.96 10.45 1.54
± ± ± ± ± ± ± ±
0.92 0.78 1.16 0.71 0.00 0.69 0.68 3.25
appears that the auto-mechanics seem to stay longer on the job compared to any other group. The number of hours spent per day in the working place for the various groups of artisans is also depicted in Table 2. With the exception of drivers and spare parts dealers, majority of the artisans spend an average of 11 h/day in their workshops. The petrol station attendants on the other hand spend an average of 14.3 h/day with their nozzle. The mean blood lead concentrations in the different artisans before and after Vitamin C administration are depicted in Table 3. Before the Vitamin C administration, lead levels in the blood of the artisans were significantly higher than that of control (p < 0.001). Of the artisans, the auto-electricians had the highest blood lead level of 48.90 ± 19.11 g/dl, a value which was three times higher than that of the control. The welders on the other hand, had the lowest value of 27.00 ± 1.05 g/dl which was still 1.7 times that of the control subjects. At the end of the 2-week Table 3 Mean blood lead concentrations of the subjects Subject
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
Blood lead Before ascorbic acid
After ascorbic acid
Percentage reduction after ascorbic acid
15.78 ± 2.84 a 43.98 ± 10.54 e 48.90 ± 19.11 f 41.13 ± 11.55 d 40.99 ± 7.00 d 36.10 ± 9.64 c 35.99 ± 11.08 c 42.53 ± 5.90 d
12.49 ± 2.31 a 22.68 ± 5.18 d 22.56 ± 3.98 d 25.00 ± 7.07 e – 19.40 ± 3.07 c 23.59 ± 6.15 d 16.29 ± 1.95 b
20.55 ± 7.94 48.03 ± 13.16 49.36 ± 16.27 34.20 ± 35.67 – 49.78 ± 6.79 42.91 ± 10.61 60.84 ± 8.55
31.61 ± 7.78 c 35.79 ± 6.30 c
12.10 ± 2.13 a 20.01 ± 5.32 d
57.71 ± 21.74 44.53 ± 5.09
27.00 ± 1.05 b
12.73 ± 0.03 a
52.81 ± 1.74
Values are mean ± S.D. and are expressed in g/dl. Values in a column not having any letter (a–f) in common are significantly different from each other (p < 0.001).
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Table 4 ALAD activity in the subjects before and after ascorbic acid supplementation Subject
ALAD activity Before ascorbic acid
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
After ascorbic acid
Activity (U/l)
Percentage of control
Degree of inhibition (%)
Activity (U/l)
Percentage of control
Degree of inhibition (%)
60.98 ± 10.79 a 23.29 ± 8.05 d 17.20 ± 9.58 e 14.67 ± 10.97 e 20.30 ± 1.36 e 31.15 ± 7.91 c 21.39 ± 8.26 e 13.78 ± 7.31 e 33.05 ± 9.71 c 22.90 ± 12.64 e 39.17 ± 1.94 b
100.0 38.2 28.2 24.1 33.3 51.1 35.1 22.6 54.2 37.6 64.2
0 61.8 71.8 75.9 66.7 48.9 64.9 77.4 45.8 62.4 35.8
67.31 ± 9.73 a 35.51 ± 6.84 d 30.25 ± 5.53 f 30.66 ± 2.50 f – 42.68 ± 7.08 c 35.41 ± 5.49 d 26.55 ± 9.83 f 48.87 ± 1.28 b 34.25 ± 8.39 e 49.00 ± 2.47 b
100.0 52.8 44.9 45.6 – 63.4 52.6 39.4 72.6 50.9 72.8
0 37.2 55.1 54.4 – 36.6 47.4 60.6 27.4 49.1 27.2
Activity values are mean ± S.D. and are expressed in U/l while other values are expressed in percentages relative to control. Values in a column not having any letter (a–f) in common are significantly different from each other (p < 0.001).
ascorbic acid supplementation, there was a significant reduction (p < 0.001) in the blood lead levels of the subjects. While there was a 21% reduction in the blood lead levels of the control, it ranged from 34% in the battery chargers to 61% in the petrol station attendants (Table 3). Table 4 shows the mean activities of ALAD observed in the different artisans before and after ascorbic acid regimen and ALAD activities in the artisans expressed as percent of control and percent inhibition. ALAD was significantly (p < 0.001) inhibited to varying extents in the artisans. The lowest activity was observed in the petrol station attendants. When expressed in terms of activity of control, ALAD activity in the petrol station attendants was just 23% of control, indicating an inhibition of 77%. In contrast however, a 36% inhibition of ALAD activity was observed in the welders. Administration of Vitamin C resulted in a significant (p < 0.001) increase in ALAD activity in the artisans. In the auto-electricians, battery chargers and petrol station attendants, there was a 2-fold increase in the activity of ALAD. In the other groups of artisans, increases ranging
between 1.3 and 1.7-fold were observed. Even though blood lead levels comparable to control values were observed in the upholsterers, welders and petrol station attendants at the end of the 2-week ascorbic acid therapy (Table 3), ALAD activity did not fully return to control values. In the petrol station attendants ALAD activity was just 40% of control value, while in the welders and upholsterers, it was 73% of control. EPP concentrations in the subjects are depicted in Table 5. Before the ascorbic acid supplementation, the mean EPP levels observed in the artisans were significantly higher than that of control (p < 0.001). The upholsterers had the highest value of 241.57 ± 89.27 g/100 ml of RBC, followed by the petrol station attendants with 204.23 ± 95.24 g/100 ml RBC. Quantitatively, mean EPP concentrations observed in the lead exposed subjects were between 2.23 and 3.21 times higher than the controls. The 2-week Vitamin C supplementation also resulted in a significant decrease in the EPP levels of all the subjects (p < 0.001). While the reduction amounted to 50% in controls, the reduction ranged between 45 and 69% for the artisans. Quantitatively, the highest reduction in EPP
Table 5 Erythrocyte protoporphyrin (EPP) in the subjects before and after ascorbic acid supplementation Subject
EPP concentration Before ascorbic acid
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
75.14 170.54 189.61 177.82 180.41 171.64 175.99 204.23 241.57 167.79 174.20
± ± ± ± ± ± ± ± ± ± ±
12.32 a 42.10 b 66.19 b 14.74 b 5.87 b 18.23 b 38.90 b 95.24 c 89.27 d 23.55 b 2.91 b
After ascorbic acid
Percentage reduction after ascorbic acid
37.32 ± 87.95 ± 78.66 ± 65.88 ± – 74.77 ± 91.53 ± 89.41 ± 141.63 ± 77.79 ± 53.70 ±
49.60 ± 7.37 50.05 ± 13.62 55.48 ± 11.94 62.75 ± 5.00 – 55.74 ± 10.12 48.96 ± 11.72 52.14 ± 11.23 45.83 ± 20.12 52.38 ± 17.93 69.18 ± 0.75
4.65 a 20.61 c 10.88 c 3.39 a 11.85 b 41.91 c 14.53 c 104.32 d 18.88 b 2.21 a
Values are mean ± S.D. and are expressed in g/100 ml of RBC. Values in a column not having any letter (a–d) in common are significantly different from each other (p < 0.001).
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Table 6 Correlations between blood lead levels and lead-related effects Parameter
ALAD activity vs. blood lead ALAD activity vs. EPP Blood lead vs. EPP
Before ascorbic acid
After ascorbic acid
Correlation equation
Correlation coefficient (r)
P value
y = −0.68442x + 53.88 y = −0.1194x + 47.77 y = 0.08661x + 24.22
−0.631 −0.461 0.366
3.0 × 10−12 y = −1.3434x + 67.13 1.0 × 10−6 y = −0.1136x + 48.05 4.0 × 10−5 y = 0.02260x + 18.51
was observed in the welders. At the end of the 2-week Vitamin C regimen, the highest EPP levels were still observed in the upholsterers. Both before and after Vitamin C supplementation, significant correlations (p < 0.001) were observed between blood lead levels and lead related effects (Table 6). Both before and after ascorbic acid supplementation, ALAD activity in the artisans was significantly negatively correlated with blood lead levels. Similar results were obtained for ALAD activity and EPP. Even though the correlations between EPP and its ALAD activity were not so high, they were still statistically significant (before ascorbic acid r = −0.461, p = 1 × 10−6 ; after ascorbic acid r = −0.283, p = 0.005). In contrast however, blood lead levels and their EPP concentrations were positively correlated. Among the subjects, it was observed that the correlations of EPP with blood lead was slightly poor compared to that of ALAD activity and blood lead. At the end of the 2-week ascorbic acid regimen, the correlation between blood lead and EPP was not statistically significant.
4. Discussion The results of this investigation demonstrate that in addition to the petrol station attendants and auto-mechanics who have been shown to be occupationally exposed to lead (Ademuyiwa et al., 2002; Dosumu et al., 2005), other artisans in Abeokuta are indeed occupationally exposed to lead and this was reflected in the significantly high levels of blood lead in comparison with the controls. The lead concentrations in their blood varied from 20.06 to 80.74 g/dl. The most probable source of lead exposure seems to be the leaded gasoline still sold in Nigeria. High lead concentrations in air and soil in urban areas like Abeokuta, have been attributed to ever increasing automobiles, especially using leaded gasoline (Nriagu et al., 1996). Uncontrolled burning of refuse, wood, agricultural wastes, paper products, discarded tyres and battery casings, also contributes to this environmental lead loading. While the general population is presented with lead from the environment, the occupationally exposed subjects are presented with lead from occupational habits. It is the practice among the artisans in Nigeria to suck/siphon petrol with the mouth and sprinkle/spray it upon carburettors, car engines, kick starters and other parts of the car and engine when washing them until the petrol in their mouth is exhausted. Through this, they either inhale or ingest the
Correlation equation
Correlation coefficient (r)
P value
−0.586 −0.283 0.143
3.0 × 10−9 0.005 0.099
lead that is present in the gasoline. From the questionnaire administered, the auto-mechanics do this (on average, and depending on the number of carburettors and engines washed per day), six times a day, the painters, drivers and spare parts/oil sellers, once in a day, the auto-electricians once a week and the panel beaters twice in a month. While the battery chargers are exposed to lead by tasting battery water (which contains a considerable amount of lead) seven times in a day, the petrol station attendants might be presented with lead from fumes of lead inhaled from the leaded-gasoline when filling car tanks. The welders on the other hand might be presented with lead from welding fumes. Dusts, arising from vehicle seats which they mend, might be the source of lead exposure for the upholsterers. Although the contributions of other environmental sources cannot be ruled out, the percentage contribution of these towards the total lead burden of the indidvidual artisan might be difficult to evaluate. Over the years, several attempts have been made to relate blood lead levels to adverse health effects. Even though the concentration of lead in blood reflects a dynamic equilibrium between exposure, absorption, distribution and elimination of lead (UNEP/WHO, 1985), assessment of risks to health of this blood lead concentration is usually carried out by measuring some biochemical end-points which lead specifically affects. A lot of studies have been carried out using the biological response of the levels of metabolites or the activity of enzymes related to heme biosynthesis and depletion of some essential metals in the body to monitor lead exposure and toxicity (Chiba et al., 1996; Dosumu et al., 2005; Mehdi et al., 2000; Onunkwor et al., 2004; Sakai and Morita, 1996). The artisans in this study were monitored for hematotoxic effects of lead by determining the activity of ALAD and the concentration of EPP in their blood and these were related to the degree of lead absorption. The present findings indicate a statistically significant inhibition of between 36 and 77% in the activity of ALAD and an increase in the EPP in these artisans and these were negatively correlated with blood lead levels. These results confirm the inhibiting effect of lead on ALAD and that this inhibition increases with increasing blood lead levels. Numerous animal models and previous epidemiologic studies among workers and populations of various ages who were moderately or highly exposed to lead, have reported negative relationships between ALAD activity and lead exposure (Campagna et al., 1999; Chiba et al., 1996; Prpic-Majic and Telisman, 1990; Tandon et al., 2001; Tomokuni and Ogata, 1976). Chiba and
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his colleagues (Chiba et al., 1996) even reported an inhibition rate between 23 and 71% in ALAD in those who were occupationally exposed. Even though this is the first report of such a study in Nigeria, the findings of this present study are in good agreement with the observations of these other workers. The levels of EPP reflect the toxic effects of lead on the erythrocytic enzyme ferrochelatase (Staudinger and Roth, 1998). EPP levels usually begin to rise in adults when the blood lead level exceeds 20 g/dl. Once elevated, EPP levels tend to remain above background level for several months (Staudinger and Roth, 1998). EPP is formed in the mitochondria during differentiation of erythrocytes in the bone marrow and the conversion of protoporphyrin to heme requires the insertion of iron into the protoporphyrin ring in the mitochondria. Lead has been shown to inhibit this insertion of iron into the protoporphyrin ring, a reaction catalysed by ferrochelatase (Goyer, 1991; Staudinger and Roth, 1998). Since mitochondria are lost when the red cell matures, EPP in circulating red cells represents that which was present during the maturation of the red cells in the bone marrow (Goyer, 1991). The increased levels of EPP observed in the circulating erythrocytes of these artisans reflect primarily the biological effects of lead in their bone marrow, thus suggesting an incomplete maturation of the red blood cells which eventually is observed as shortened erythrocyte life span. Administration of a daily dose of 500 mg ascorbic acid for 2 weeks to these artisans not only resulted in a significant reduction in blood lead levels, the activity of ALAD was also significantly increased. In the petrol station attendants and battery chargers, there was a double-fold increase in ALAD activity. Similar observations were made for other subjects. In addition, increased levels of EPP observed before were significantly reduced in all the subjects as a result of the ascorbic acid supplementation, suggesting indirectly that the activity of ferrochelatase is restored. About a decade ago, some animal studies indicate that orally administered ascorbic acid may chelate lead and decrease the risk of the toxic effects of the metal (Dalley et al., 1990). Earlier studies in rats demonstrated that ascorbic acid decreased the intestinal absorption of lead and increased the renal clearance of the metal (Morton et al., 1985; Niazi et al., 1982). In another study (Onunkwor et al., 2004), it was demonstrated that alterations induced in the levels of GSH, hemoglobin, ALA and calcium by lead were reversed by ascorbic acid. Furthermore, ascorbic acid supplementation resulted not only in reduced blood lead levels, urinary excretion of lead also increased (Calabrese et al., 1987; Dawson et al., 1999; Lauwerys et al., 1983; Onunkwor et al., 2004). These observations, together with the findings of the present investigation, confirm the protective effect of ascorbic acid in lead-induced toxicity. ALAD enzyme is a metalloenzyme which requires Zn2+ and reduced thiol (SH) groups for maximal catalytic activity (Campagna et al., 1999; Dent et al., 1990). It has eight identical subunits and eight binding sites for Zn2+ which are
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important for its function (Campagna et al., 1999; Dent et al., 1990). The binding of the first four Zn2+ might prevent the sulphydryl (SH) function oxidation of ALAD active site (Jaffe et al., 1984), while the fixation of the four other Zn2+ might help balance the ALAD structure (Dent et al., 1990). The presence of sulphydryl groups is necessary for the binding of Zn2+ to the enzyme. Ionic lead (Pb2+ ) has been shown to replace Zn2+ on ALAD binding sites (Bergdahl et al., 1998; Jaffe et al., 2001; Simons, 1995) which leads to bridging of the SH functions to S–S and eventually to ALAD inhibition. Lead has also been shown to reduce the absorption of zinc (Flora, 1991). The protective action of ascorbic acid on ALAD might be due to removal of Pb2+ from the active site by forming a complex with it (Dalley et al., 1990; Dhawan et al., 1988; Flora and Tandon, 1986; Onunkwor et al., 2004) and restoring the SH function of the enzyme by making reduced thiol groups available (Onunkwor et al., 2004). On restoration of the SH function of the enzyme, a channel is then opened through which Zn2+ could be made available for binding to the zinc-binding sites on the enzyme (Gonz´alez et al., 1987; Jaffe et al., 2001). A similar hypothesis for ferrochelatase (removal of Pb2+ from the active site and making iron available) might be responsible for the reduced EPP observed in the artisans as a result of ascorbic acid supplementation (Harper, 1979). The increased urinary excretion of lead observed in lead-exposed workers as a result of ascorbic acid supplementation might also provide an indirect evidence for complex formation between ascorbic acid and lead (Dawson et al., 1999; Onunkwor et al., 2004). Further studies are on in this laboratory to establish whether other lead-related effects will respond positively to the ameliorative effects of ascorbic acid. In all cases of suspected lead intoxication in adults, the first step in management should be the removal of the individual from the exposure and thereafter institution of chelation therapy (Fischbein, 1992; Landrigan, 1994; Lewis, 1990). The most commonly employed antidotes for lead poisoning are calcium disodium ethylene diamine tetra-acetic acid (CaNa2 EDTA) and succimer (Dawson et al., 1999; Gurer and Ercal, 2000; Staudinger and Roth, 1998). It should be noted that these chelating agents themselves have serious side effects which in itself represents a risk apart from the lead toxicity (Gurer and Ercal, 2000). Ascorbic acid appears to offer an advantage in that the lead-exposed individual does not have to be removed from the source of lead exposure and be hospitalised (Onunkwor et al., 2004). In addition, ascorbic acid can be administered orally and is readily available and cheap. In conclusion, the findings of this study indicate that ALAD activity is inhibited to varying extents in artisans in Abeokuta, Nigeria, as a result of exposure to lead and that ascorbic acid reverses lead-induced inhibition of this enzyme. Furthermore, lead-induced shortening of erythrocyte life span appears to be attenuated by Vitamin C. Further studies are necessary in order to investigate what other advantages ascorbic acid offers compared to the common chelating
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agents like CaNa2 EDTA and succimer in the treatment of lead poisoning.
Acknowledgements The authors are grateful to the heads of the two mechanic workshops and the other subjects in the workshops who participated in the study. Our thanks are also due to the medical and nursing teams of University of Agriculture Health Centre, Abeokuta, Nigeria, for their support in this study. The technical assistance of Mrs. J.O. Adebawa is also highly appreciated.
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Reversal of aminolevulinic acid dehydratase (ALAD) inhibition and reduction of erythrocyte protoporphyrin levels by Vitamin C in occupational lead exposure in Abeokuta, Nigeria O. Ademuyiwa a,∗ , R.N. Ugbaja a , D.A. Ojo b , A.O. Owoigbe a , S.E. Adeokun a a b
Department of Biochemistry, University of Agriculture, Abeokuta, Nigeria Department of Microbiology, University of Agriculture, Abeokuta, Nigeria Received 12 December 2004; accepted 20 April 2005 Available online 27 June 2005
Abstract In order to investigate the toxic effects of lead during occupational exposure to this metal, the activity of aminolevulinic acid dehydratase (ALAD) and the concentrations of erythrocyte protoporphyrin (EPP) were determined in blood of various artisans in Abeokuta, Nigeria and these were related to blood lead levels. ALAD activity in the artisans was inhibited to varying extents. ALAD activity was inhibited to the tune of 77% in petrol station attendants while the lowest inhibition of 36% was obtained in the welders. EPP was also significantly increased in the artisans (p < 0.001). The highest EPP level of 241.57 ± 89.27 g/100 ml of red blood cell was observed in upholsterers. A significant (p < 0.001) negative correlation was observed between ALAD activity and blood lead levels on one hand (r = −0.631) and between ALAD activity and EPP on the other hand (r = −0.461). Administration of a daily dose of 500 mg ascorbic acid for 2 weeks reversed the lead-induced inhibition of ALAD. Increased EPP levels observed in the artisans also responded positively to the ascorbic acid supplementation. A significant reduction (p < 0.001) in blood lead was also observed in the artisans at the end of the 2-week ascorbic acid therapy. Our findings indicate that ascorbic acid may offer more advantages over the conventional agents for the treatment of lead poisoning, especially in cases where the subjects cannot be removed from the source of lead exposure. © 2005 Elsevier B.V. All rights reserved. Keywords: ALAD inhibition; Erythrocyte protoporphyrin; Artisans in Nigeria; Reversal; Ascorbic acid
1. Introduction Lead, the most ubiquitous toxic metal found in the earth’s crust, has been used by human beings for thousands of years and it is still one of the most widely used metals in industries (Pande and Flora, 2002). The health hazards of lead and its compounds have been the focus of scientific attention in many countries of the world (Ademuyiwa et al., 2002; Canfield et al., 2003; Lalor et al., 2001; Nriagu et al., 1997; Onunkwor et al., 2004). Numerous studies have demonstrated that lead is a common environmental and industrial pollutant that affects ∗
Corresponding author. Tel.: +234 803 357 5238. E-mail address: [email protected] (O. Ademuyiwa).
1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2005.04.002
virtually every system in the body (Ademuyiwa et al., 2002; Albalak et al., 2003; Canfield et al., 2003; Kentner et al., 1994; Onunkwor et al., 2004; Sakai, 2000). Neurobehavioral, hematologic, nephrotoxic and reproductive effects of lead have been observed in humans and other animals (Elinder et al., 1988; Goyer, 1991; Needleman et al., 1990; Rice, 1996). The immune system is not spared from the toxic effects of lead (Basaran and Undeger, 2000). One of the best known toxic effects of lead is its interference with heme biosynthesis (Alexander et al., 1998; Flora et al., 2003; Makino et al., 2000). Elevation of delta-aminolevulinic acid (ALA) level, due to inhibition of delta-aminolevulinic acid dehydratase (ALAD) enzyme, followed by elevation of protoporphyrins in erythrocytes and increased urinary excretion of ALA and coproporphyrin, are
O. Ademuyiwa et al. / Environmental Toxicology and Pharmacology 20 (2005) 404–411
early effects associated with increasing lead concentration in soft tissues (Alexander et al., 1998; Flora et al., 2003; Sakai, 2000). Elevation of ALA results in overproduction of active oxygen species (Gurer et al., 1999; Neal et al., 1997). Because of its high sensitivity to lead, inhibition of ALAD has been used as an index of environmental lead pollution (Campagna et al., 1999; Chalevelakis et al., 1995; Flora et al., 2003; Tandon et al., 2001). Lead exposure studies (both environmental and occupational) is a well-trodden subject in developed countries (Canfield et al., 2003; EPA, 1986; Goyer, 1993; Meyer et al., 1998; Pirkle et al., 1994; P¨onka, 1998; Rice, 1996; Stromberg et al., 1995). In contrast however, such information is still relatively scarce in developing countries like Nigeria. In previous studies, elevated lead levels were observed in petrol station attendants and auto-mechanics in Abeokuta, Nigeria (Ademuyiwa et al., 2002; Dosumu et al., 2005; Onunkwor et al., 2004). Information about lead levels in other artisans in Nigeria is lacking. In addition, no study has been undertaken in Nigeria to determine the extent of inhibition of ALAD in those who are occupationally exposed to lead. For these reasons, ALAD activity and the concentration of erythrocyte protoporphyrin (EPP) were determined in blood of different groups of artisans in Abeokuta, Nigeria and these were related to blood lead levels. Petrol station attendants and auto-mechanics were included in the study in order to compare their ALAD levels with other artisans since they have been shown to be occupationally exposed to lead (Ademuyiwa et al., 2002; Dosumu et al., 2005; Onunkwor et al., 2004). The hypothesis was also tested whether ascorbic acid would remove lead-induced inhibition of ALAD.
2. Subjects and methods 2.1. Study population A total of 110 male subjects participated in the study. Control subjects were made up of staff and students of University of Agriculture, Abeokuta, Nigeria, while leadexposed subjects comprised of different artisans located in two mechanic workshops in the southern and northern parts of the city of Abeokuta, Nigeria. It is typical of mechanic workshops in Nigeria to find other groups of artisans located in these workshops in addition to the auto-mechanics, thereby complementing each other’s services. In these workshops, there is always a preponderance of auto-mechanics. After explaining the objectives of the study to them, a total of 92 artisans, including 50 auto-mechanics, consented to participate in the study. Seven male petrol station attendants from a petrol station in the southern part of the city were also included among the occupationally-exposed subjects. A careful history of their dietary habit and their job experience was taken. Table 1 summarises the study population.
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Table 1 Study population Subject
Number
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
11 50 8 2 2 7 15 7 4 2 2
All the subjects studied were non-smokers. Six of the auto-mechanics were alcoholics, taking one bottle of beer per day; five of the auto-mechanics, two panel beaters, two petrol station attendants and one auto-electrician, were occasional alcoholics, taking between one and two bottles of beer in 2 months. 2.2. Supplementation regimen The ascorbic acid (Mopson Pharmaceuticals, Lagos, Nigeria) was given as a daily dose of 500 mg ascorbic acid for 2 weeks to each subject. During ascorbic acid supplementation period, occasional visits were paid to the subjects to ensure compliance with the regimental protocols. 2.3. Sampling Blood samples were collected before and after ascorbic acid supplementation. Venous blood samples (about 10 ml) were collected from each subject by a trained medical laboratory staff into heparinised tubes. The blood samples were stored in a cooler box and then transferred to the laboratory for analysis. The two drivers were away on a journey on the day the second blood samples were to be collected; as such, no values were reported for them. 2.4. Lead analysis Lead was determined in whole blood by atomic absorption spectrometry (Buck Scientific AAS Model 210, Connecticut, USA). 1 ml of whole blood was digested with concentrated nitric acid and digests brought to a constant volume. External standardisation for lead was done by analysing a certified Spex Lead Standard (Spex Industries Inc., Edison, New Jersey, USA) along with the samples. Procedural accuracy of the lead analyses was also evaluated by spiking control samples with the Spex Lead Standard. In all cases, two determinations were made for each sample and the mean lead recovery rate of 98.37 ± 4.57% was obtained for all the analyses.
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2.5. Determination of ALAD activity
Table 2 Some demographic characteristics of the subjects
The activity of ALAD was also determined in whole blood less than 24 h after blood collection using the European Standardised Method described by Berlin and Schaller (1974). 0.2 ml of heparinised blood was mixed with 1.3 ml of distilled water and incubated for 10 min at 37 ◦ C. 1 ml of standard ALA was then added and the tubes were further incubated for 1 h. The reaction was stopped by adding 1 ml trichloroacetic acid (TCA) containing mercuric chloride (HgCl2 ). To 1 ml of the supernatant, 1 ml of Ehrlich’s reagent was added and the absorbance was read at 555 nm after 5 min.
Subject
Age in years
Job experience in years
Hours spent per day in workshop
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
26.00 ± 6.65 28.02 ± 10.72 32.00 ± 10.17 27.00 ± 7.07 39.50 ± 12.02 34.00 ± 19.56 29.53 ± 13.03 28.71 ± 3.99
– 11.64 13.25 7.50 4.00 15.71 12.47 3.57
8.64 11.62 11.25 11.50 10.00 11.14 11.20 14.29
29.25 ± 8.96 30.50 ± 0.71
12.38 ± 11.46 6.50 ± 2.12
11.25 ± 1.50 10.50 ± 2.12
37.50 ± 3.54
25.50 ± 0.71
11.50 ± 0.71
2.6. Determination of erythrocyte protoporphyrin (EPP) EPP was determined in whole blood using the method of Heller et al. (1971). Briefly, 2 ml of heparinised blood was mixed with 2 ml of acetone:ethylacetate mixture (1:9, v/v) in a 10 ml centrifuge tube. The mixture was then vortexed vigorously for about 1 min. Four millilitres of formic acid:diethylether mixture (1:9, v/v) was then added and the mixture vortexed again for another 1 min. This was then centrifuged at 5000 rpm for 5 min. The supernatant was decanted into a 10 ml graduated centrifuge tube and the precipitate reextracted with a second 4 ml formic acid:diethylether mixture. To the combined supernatants, 2 ml 1.5 M HCl was added and this was vortexed vigorously for 30 s. The volume of the (lower) HCl layer was recorded. The HCl layer was then transferred into a small test tube with a long fine tip pipet. The absorbance of this solution was then read at 380, 407 and 430 nm against 1.5 M HCl blank with a Jenway 6405 UV/Visible spectrophotometer (Jenway Ltd., Felsted, Dunmow, Essex, UK). 2.7. Statistical protocol Results are expressed as mean ± S.D. One-way analysis of variance (ANOVA) followed by the LSD (least significant difference) test were used to analyse the results with p < 0.05 (Sachs, 1983). The relationships between ALAD activities and lead exposure measurements on one hand; and between ALAD activities and EPP concentrations on the other hand, both before and after the 2-week ascorbic acid supplementation, were analysed using Pearson correlations (Sachs, 1983). The relationships between blood lead levels and EPP both before and after ascorbic acid supplementation were also analysed using Pearson correlations (Sachs, 1983).
3. Results Some demographic characteristics of the subjects are depicted in Table 2. Their ages ranged between 18 and 66 years. The years of experience on the job for each group shows a wide variation, ranging from 0.7 to 53 in the automechanics and 1.5 to 5 in the petrol station attendants. It
± ± ± ± ± ± ±
10.92 11.18 0.71 2.83 19.96 10.45 1.54
± ± ± ± ± ± ± ±
0.92 0.78 1.16 0.71 0.00 0.69 0.68 3.25
appears that the auto-mechanics seem to stay longer on the job compared to any other group. The number of hours spent per day in the working place for the various groups of artisans is also depicted in Table 2. With the exception of drivers and spare parts dealers, majority of the artisans spend an average of 11 h/day in their workshops. The petrol station attendants on the other hand spend an average of 14.3 h/day with their nozzle. The mean blood lead concentrations in the different artisans before and after Vitamin C administration are depicted in Table 3. Before the Vitamin C administration, lead levels in the blood of the artisans were significantly higher than that of control (p < 0.001). Of the artisans, the auto-electricians had the highest blood lead level of 48.90 ± 19.11 g/dl, a value which was three times higher than that of the control. The welders on the other hand, had the lowest value of 27.00 ± 1.05 g/dl which was still 1.7 times that of the control subjects. At the end of the 2-week Table 3 Mean blood lead concentrations of the subjects Subject
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
Blood lead Before ascorbic acid
After ascorbic acid
Percentage reduction after ascorbic acid
15.78 ± 2.84 a 43.98 ± 10.54 e 48.90 ± 19.11 f 41.13 ± 11.55 d 40.99 ± 7.00 d 36.10 ± 9.64 c 35.99 ± 11.08 c 42.53 ± 5.90 d
12.49 ± 2.31 a 22.68 ± 5.18 d 22.56 ± 3.98 d 25.00 ± 7.07 e – 19.40 ± 3.07 c 23.59 ± 6.15 d 16.29 ± 1.95 b
20.55 ± 7.94 48.03 ± 13.16 49.36 ± 16.27 34.20 ± 35.67 – 49.78 ± 6.79 42.91 ± 10.61 60.84 ± 8.55
31.61 ± 7.78 c 35.79 ± 6.30 c
12.10 ± 2.13 a 20.01 ± 5.32 d
57.71 ± 21.74 44.53 ± 5.09
27.00 ± 1.05 b
12.73 ± 0.03 a
52.81 ± 1.74
Values are mean ± S.D. and are expressed in g/dl. Values in a column not having any letter (a–f) in common are significantly different from each other (p < 0.001).
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Table 4 ALAD activity in the subjects before and after ascorbic acid supplementation Subject
ALAD activity Before ascorbic acid
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
After ascorbic acid
Activity (U/l)
Percentage of control
Degree of inhibition (%)
Activity (U/l)
Percentage of control
Degree of inhibition (%)
60.98 ± 10.79 a 23.29 ± 8.05 d 17.20 ± 9.58 e 14.67 ± 10.97 e 20.30 ± 1.36 e 31.15 ± 7.91 c 21.39 ± 8.26 e 13.78 ± 7.31 e 33.05 ± 9.71 c 22.90 ± 12.64 e 39.17 ± 1.94 b
100.0 38.2 28.2 24.1 33.3 51.1 35.1 22.6 54.2 37.6 64.2
0 61.8 71.8 75.9 66.7 48.9 64.9 77.4 45.8 62.4 35.8
67.31 ± 9.73 a 35.51 ± 6.84 d 30.25 ± 5.53 f 30.66 ± 2.50 f – 42.68 ± 7.08 c 35.41 ± 5.49 d 26.55 ± 9.83 f 48.87 ± 1.28 b 34.25 ± 8.39 e 49.00 ± 2.47 b
100.0 52.8 44.9 45.6 – 63.4 52.6 39.4 72.6 50.9 72.8
0 37.2 55.1 54.4 – 36.6 47.4 60.6 27.4 49.1 27.2
Activity values are mean ± S.D. and are expressed in U/l while other values are expressed in percentages relative to control. Values in a column not having any letter (a–f) in common are significantly different from each other (p < 0.001).
ascorbic acid supplementation, there was a significant reduction (p < 0.001) in the blood lead levels of the subjects. While there was a 21% reduction in the blood lead levels of the control, it ranged from 34% in the battery chargers to 61% in the petrol station attendants (Table 3). Table 4 shows the mean activities of ALAD observed in the different artisans before and after ascorbic acid regimen and ALAD activities in the artisans expressed as percent of control and percent inhibition. ALAD was significantly (p < 0.001) inhibited to varying extents in the artisans. The lowest activity was observed in the petrol station attendants. When expressed in terms of activity of control, ALAD activity in the petrol station attendants was just 23% of control, indicating an inhibition of 77%. In contrast however, a 36% inhibition of ALAD activity was observed in the welders. Administration of Vitamin C resulted in a significant (p < 0.001) increase in ALAD activity in the artisans. In the auto-electricians, battery chargers and petrol station attendants, there was a 2-fold increase in the activity of ALAD. In the other groups of artisans, increases ranging
between 1.3 and 1.7-fold were observed. Even though blood lead levels comparable to control values were observed in the upholsterers, welders and petrol station attendants at the end of the 2-week ascorbic acid therapy (Table 3), ALAD activity did not fully return to control values. In the petrol station attendants ALAD activity was just 40% of control value, while in the welders and upholsterers, it was 73% of control. EPP concentrations in the subjects are depicted in Table 5. Before the ascorbic acid supplementation, the mean EPP levels observed in the artisans were significantly higher than that of control (p < 0.001). The upholsterers had the highest value of 241.57 ± 89.27 g/100 ml of RBC, followed by the petrol station attendants with 204.23 ± 95.24 g/100 ml RBC. Quantitatively, mean EPP concentrations observed in the lead exposed subjects were between 2.23 and 3.21 times higher than the controls. The 2-week Vitamin C supplementation also resulted in a significant decrease in the EPP levels of all the subjects (p < 0.001). While the reduction amounted to 50% in controls, the reduction ranged between 45 and 69% for the artisans. Quantitatively, the highest reduction in EPP
Table 5 Erythrocyte protoporphyrin (EPP) in the subjects before and after ascorbic acid supplementation Subject
EPP concentration Before ascorbic acid
Control Auto-mechanics Auto-electricians Battery chargers Drivers Painters (car) Panel beaters Petrol station attendants Upholsterers Spare parts/oil sellers Welders
75.14 170.54 189.61 177.82 180.41 171.64 175.99 204.23 241.57 167.79 174.20
± ± ± ± ± ± ± ± ± ± ±
12.32 a 42.10 b 66.19 b 14.74 b 5.87 b 18.23 b 38.90 b 95.24 c 89.27 d 23.55 b 2.91 b
After ascorbic acid
Percentage reduction after ascorbic acid
37.32 ± 87.95 ± 78.66 ± 65.88 ± – 74.77 ± 91.53 ± 89.41 ± 141.63 ± 77.79 ± 53.70 ±
49.60 ± 7.37 50.05 ± 13.62 55.48 ± 11.94 62.75 ± 5.00 – 55.74 ± 10.12 48.96 ± 11.72 52.14 ± 11.23 45.83 ± 20.12 52.38 ± 17.93 69.18 ± 0.75
4.65 a 20.61 c 10.88 c 3.39 a 11.85 b 41.91 c 14.53 c 104.32 d 18.88 b 2.21 a
Values are mean ± S.D. and are expressed in g/100 ml of RBC. Values in a column not having any letter (a–d) in common are significantly different from each other (p < 0.001).
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Table 6 Correlations between blood lead levels and lead-related effects Parameter
ALAD activity vs. blood lead ALAD activity vs. EPP Blood lead vs. EPP
Before ascorbic acid
After ascorbic acid
Correlation equation
Correlation coefficient (r)
P value
y = −0.68442x + 53.88 y = −0.1194x + 47.77 y = 0.08661x + 24.22
−0.631 −0.461 0.366
3.0 × 10−12 y = −1.3434x + 67.13 1.0 × 10−6 y = −0.1136x + 48.05 4.0 × 10−5 y = 0.02260x + 18.51
was observed in the welders. At the end of the 2-week Vitamin C regimen, the highest EPP levels were still observed in the upholsterers. Both before and after Vitamin C supplementation, significant correlations (p < 0.001) were observed between blood lead levels and lead related effects (Table 6). Both before and after ascorbic acid supplementation, ALAD activity in the artisans was significantly negatively correlated with blood lead levels. Similar results were obtained for ALAD activity and EPP. Even though the correlations between EPP and its ALAD activity were not so high, they were still statistically significant (before ascorbic acid r = −0.461, p = 1 × 10−6 ; after ascorbic acid r = −0.283, p = 0.005). In contrast however, blood lead levels and their EPP concentrations were positively correlated. Among the subjects, it was observed that the correlations of EPP with blood lead was slightly poor compared to that of ALAD activity and blood lead. At the end of the 2-week ascorbic acid regimen, the correlation between blood lead and EPP was not statistically significant.
4. Discussion The results of this investigation demonstrate that in addition to the petrol station attendants and auto-mechanics who have been shown to be occupationally exposed to lead (Ademuyiwa et al., 2002; Dosumu et al., 2005), other artisans in Abeokuta are indeed occupationally exposed to lead and this was reflected in the significantly high levels of blood lead in comparison with the controls. The lead concentrations in their blood varied from 20.06 to 80.74 g/dl. The most probable source of lead exposure seems to be the leaded gasoline still sold in Nigeria. High lead concentrations in air and soil in urban areas like Abeokuta, have been attributed to ever increasing automobiles, especially using leaded gasoline (Nriagu et al., 1996). Uncontrolled burning of refuse, wood, agricultural wastes, paper products, discarded tyres and battery casings, also contributes to this environmental lead loading. While the general population is presented with lead from the environment, the occupationally exposed subjects are presented with lead from occupational habits. It is the practice among the artisans in Nigeria to suck/siphon petrol with the mouth and sprinkle/spray it upon carburettors, car engines, kick starters and other parts of the car and engine when washing them until the petrol in their mouth is exhausted. Through this, they either inhale or ingest the
Correlation equation
Correlation coefficient (r)
P value
−0.586 −0.283 0.143
3.0 × 10−9 0.005 0.099
lead that is present in the gasoline. From the questionnaire administered, the auto-mechanics do this (on average, and depending on the number of carburettors and engines washed per day), six times a day, the painters, drivers and spare parts/oil sellers, once in a day, the auto-electricians once a week and the panel beaters twice in a month. While the battery chargers are exposed to lead by tasting battery water (which contains a considerable amount of lead) seven times in a day, the petrol station attendants might be presented with lead from fumes of lead inhaled from the leaded-gasoline when filling car tanks. The welders on the other hand might be presented with lead from welding fumes. Dusts, arising from vehicle seats which they mend, might be the source of lead exposure for the upholsterers. Although the contributions of other environmental sources cannot be ruled out, the percentage contribution of these towards the total lead burden of the indidvidual artisan might be difficult to evaluate. Over the years, several attempts have been made to relate blood lead levels to adverse health effects. Even though the concentration of lead in blood reflects a dynamic equilibrium between exposure, absorption, distribution and elimination of lead (UNEP/WHO, 1985), assessment of risks to health of this blood lead concentration is usually carried out by measuring some biochemical end-points which lead specifically affects. A lot of studies have been carried out using the biological response of the levels of metabolites or the activity of enzymes related to heme biosynthesis and depletion of some essential metals in the body to monitor lead exposure and toxicity (Chiba et al., 1996; Dosumu et al., 2005; Mehdi et al., 2000; Onunkwor et al., 2004; Sakai and Morita, 1996). The artisans in this study were monitored for hematotoxic effects of lead by determining the activity of ALAD and the concentration of EPP in their blood and these were related to the degree of lead absorption. The present findings indicate a statistically significant inhibition of between 36 and 77% in the activity of ALAD and an increase in the EPP in these artisans and these were negatively correlated with blood lead levels. These results confirm the inhibiting effect of lead on ALAD and that this inhibition increases with increasing blood lead levels. Numerous animal models and previous epidemiologic studies among workers and populations of various ages who were moderately or highly exposed to lead, have reported negative relationships between ALAD activity and lead exposure (Campagna et al., 1999; Chiba et al., 1996; Prpic-Majic and Telisman, 1990; Tandon et al., 2001; Tomokuni and Ogata, 1976). Chiba and
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his colleagues (Chiba et al., 1996) even reported an inhibition rate between 23 and 71% in ALAD in those who were occupationally exposed. Even though this is the first report of such a study in Nigeria, the findings of this present study are in good agreement with the observations of these other workers. The levels of EPP reflect the toxic effects of lead on the erythrocytic enzyme ferrochelatase (Staudinger and Roth, 1998). EPP levels usually begin to rise in adults when the blood lead level exceeds 20 g/dl. Once elevated, EPP levels tend to remain above background level for several months (Staudinger and Roth, 1998). EPP is formed in the mitochondria during differentiation of erythrocytes in the bone marrow and the conversion of protoporphyrin to heme requires the insertion of iron into the protoporphyrin ring in the mitochondria. Lead has been shown to inhibit this insertion of iron into the protoporphyrin ring, a reaction catalysed by ferrochelatase (Goyer, 1991; Staudinger and Roth, 1998). Since mitochondria are lost when the red cell matures, EPP in circulating red cells represents that which was present during the maturation of the red cells in the bone marrow (Goyer, 1991). The increased levels of EPP observed in the circulating erythrocytes of these artisans reflect primarily the biological effects of lead in their bone marrow, thus suggesting an incomplete maturation of the red blood cells which eventually is observed as shortened erythrocyte life span. Administration of a daily dose of 500 mg ascorbic acid for 2 weeks to these artisans not only resulted in a significant reduction in blood lead levels, the activity of ALAD was also significantly increased. In the petrol station attendants and battery chargers, there was a double-fold increase in ALAD activity. Similar observations were made for other subjects. In addition, increased levels of EPP observed before were significantly reduced in all the subjects as a result of the ascorbic acid supplementation, suggesting indirectly that the activity of ferrochelatase is restored. About a decade ago, some animal studies indicate that orally administered ascorbic acid may chelate lead and decrease the risk of the toxic effects of the metal (Dalley et al., 1990). Earlier studies in rats demonstrated that ascorbic acid decreased the intestinal absorption of lead and increased the renal clearance of the metal (Morton et al., 1985; Niazi et al., 1982). In another study (Onunkwor et al., 2004), it was demonstrated that alterations induced in the levels of GSH, hemoglobin, ALA and calcium by lead were reversed by ascorbic acid. Furthermore, ascorbic acid supplementation resulted not only in reduced blood lead levels, urinary excretion of lead also increased (Calabrese et al., 1987; Dawson et al., 1999; Lauwerys et al., 1983; Onunkwor et al., 2004). These observations, together with the findings of the present investigation, confirm the protective effect of ascorbic acid in lead-induced toxicity. ALAD enzyme is a metalloenzyme which requires Zn2+ and reduced thiol (SH) groups for maximal catalytic activity (Campagna et al., 1999; Dent et al., 1990). It has eight identical subunits and eight binding sites for Zn2+ which are
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important for its function (Campagna et al., 1999; Dent et al., 1990). The binding of the first four Zn2+ might prevent the sulphydryl (SH) function oxidation of ALAD active site (Jaffe et al., 1984), while the fixation of the four other Zn2+ might help balance the ALAD structure (Dent et al., 1990). The presence of sulphydryl groups is necessary for the binding of Zn2+ to the enzyme. Ionic lead (Pb2+ ) has been shown to replace Zn2+ on ALAD binding sites (Bergdahl et al., 1998; Jaffe et al., 2001; Simons, 1995) which leads to bridging of the SH functions to S–S and eventually to ALAD inhibition. Lead has also been shown to reduce the absorption of zinc (Flora, 1991). The protective action of ascorbic acid on ALAD might be due to removal of Pb2+ from the active site by forming a complex with it (Dalley et al., 1990; Dhawan et al., 1988; Flora and Tandon, 1986; Onunkwor et al., 2004) and restoring the SH function of the enzyme by making reduced thiol groups available (Onunkwor et al., 2004). On restoration of the SH function of the enzyme, a channel is then opened through which Zn2+ could be made available for binding to the zinc-binding sites on the enzyme (Gonz´alez et al., 1987; Jaffe et al., 2001). A similar hypothesis for ferrochelatase (removal of Pb2+ from the active site and making iron available) might be responsible for the reduced EPP observed in the artisans as a result of ascorbic acid supplementation (Harper, 1979). The increased urinary excretion of lead observed in lead-exposed workers as a result of ascorbic acid supplementation might also provide an indirect evidence for complex formation between ascorbic acid and lead (Dawson et al., 1999; Onunkwor et al., 2004). Further studies are on in this laboratory to establish whether other lead-related effects will respond positively to the ameliorative effects of ascorbic acid. In all cases of suspected lead intoxication in adults, the first step in management should be the removal of the individual from the exposure and thereafter institution of chelation therapy (Fischbein, 1992; Landrigan, 1994; Lewis, 1990). The most commonly employed antidotes for lead poisoning are calcium disodium ethylene diamine tetra-acetic acid (CaNa2 EDTA) and succimer (Dawson et al., 1999; Gurer and Ercal, 2000; Staudinger and Roth, 1998). It should be noted that these chelating agents themselves have serious side effects which in itself represents a risk apart from the lead toxicity (Gurer and Ercal, 2000). Ascorbic acid appears to offer an advantage in that the lead-exposed individual does not have to be removed from the source of lead exposure and be hospitalised (Onunkwor et al., 2004). In addition, ascorbic acid can be administered orally and is readily available and cheap. In conclusion, the findings of this study indicate that ALAD activity is inhibited to varying extents in artisans in Abeokuta, Nigeria, as a result of exposure to lead and that ascorbic acid reverses lead-induced inhibition of this enzyme. Furthermore, lead-induced shortening of erythrocyte life span appears to be attenuated by Vitamin C. Further studies are necessary in order to investigate what other advantages ascorbic acid offers compared to the common chelating
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agents like CaNa2 EDTA and succimer in the treatment of lead poisoning.
Acknowledgements The authors are grateful to the heads of the two mechanic workshops and the other subjects in the workshops who participated in the study. Our thanks are also due to the medical and nursing teams of University of Agriculture Health Centre, Abeokuta, Nigeria, for their support in this study. The technical assistance of Mrs. J.O. Adebawa is also highly appreciated.
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