Delta-Aminolevulinic acid dehydratase enzyme activity and susceptibility to lead toxicity in Uganda’s urban children [version 1; peer review: awaiting peer review]

Background: Rapid industrialization, urbanization, and population explosion in sub-Saharan Africa escalate environmental lead levels and subsequently blood lead levels in children. Its levels in one’s environment account for their blood lead levels. One’s susceptibility to lead toxicity is governed by nutrition status, age and genetics. This study aimed at expounding susceptibility to lead toxicity by relating blood lead levels, delta-aminolevulinic acid dehydratase (ALAD) enzyme activity, and genetic variations of proteins that code for ALAD in urban children of Uganda. Methods: A total of 198 blood samples were spectrophotometrically analysed for blood lead levels (BLL), hemoglobin (Hb) levels, and ALAD enzyme activity before DNA extraction, polymerase chain reaction, and restriction fragment length digestion for ALAD polymorphism. Results: Up to 99.5% of the total samples analyzed coded for ALAD1 allele compared to 0.05% that coded for ALAD2. There was a significant relationship between BLL, Hb status and ALAD enzyme activity in the three isozymes (ALAD1-1, ALAD1-2 and ALAD2-2) in strength of ALAD1-1 (r = 0.42, p -value = 0.02) ˂ ALAD1-2 (r = 0.62, p value = ˂ 0.001) ˂ ALAD2-2 (r = 0.67, p -value = ˂ 0.001). Conclusions: Majority of children in Uganda code for the ALAD1 allele, which is important for blood lead ions hoarding during lead toxicity. Hoarding of blood lead not only delays exposure effects but also accumulates its levels in deposit tissues and this poses adverse effects later in their lives


Introduction
Uganda like many other African countries is faced with numerous simultaneous transitions that include economic development, industrialization, population explosion and urbanization.These transitions are coming with both environmental and health changes.Population explosion is putting pressure on the environment through increased anthropogenic activities, elevated volumes of electronic wastes and this has resulted in increased volumes of toxic pollutants like Lead in both air and water bodies.Because Lead is an accumulative toxin, its increased concentration in the environment continue to pause health challenges especially to the children [1,2,3,4].Elevated environmental Lead levels have been shown to correlate with the blood Lead levels in exposed individuals [5].Childhood Lead exposure is associated with various health challenges that include lung, stomach, and bladder cancers, anemia, neuro cognitive disorders, intelligent quotient (IQ) lowering and stunted growth [4,6].Although environmental Lead pollution is preventable, in many African countries including Uganda, little attention is accorded to this problem, for example recent studies conducted in different parts of Kampala slums report elevated blood Lead levels especially among children [3,7].One's blood Levels is modulated by his age, genetics, nutritional and malaria infection status, (3,8,9].The rate of Lead ion absorption is further shown to increase with decrease in hemoglobin levels.Following its absorption, Lead sinks in red blood cells (RBCs) where it specifically binds the delta-aminolevulinic acid dehydratase (ALAD) enzyme.
It specifically catalyzes the heme formation reaction where 2 molecules of ALA are converted into monopyrrole porphobilinogen.

delta-ALA porphobilinogen +2H2O
Enzyme ALAD is rich with thiol groups, that have high affinity for Lead ions and this renders the enzyme susceptible to inhibition [10,11].It is a tetramer homodimer with eight identical subunits and located in the cytoplasm.In each of its subunits, it binds eight zinc atoms, where four zinc molecules act as catalysts whereas the remainder serve as tertiary structural stabilizers.In times of Lead burden, Lead ions displaces zinc from the enzyme's active site and inhibits its activity, resulting into heightened levels of ALA in circulation.Its susceptibility to Lead toxicity is dependent on one's age, nutritional status and the genetics [12].Accumulated levels of ALA trigger the production of reactive oxygen species (ROS) which are associated with oxidative stress.Several studies from different regions indicate varying blood Lead levels, biological, markers and even symptoms among people in the same locality.This observation is attributed to the polymorphic nature of delta-aminolevulinic acid dehydratase gene that encodes ALAD enzyme.Polymorphism of ALAD gene is reported to modulate one's susceptibility to Lead toxicity [13,14].The ALAD enzyme is encoded by a single gene on chromosome 9q34 region [15].This gene codes for two alleles i.e., ALAD-1 and ALAD-2 [16] which are codominant (Single Nucleotide Polymorphism database (dbSNP) ID: rs1800435 [17].Their expression results into a polymorphic enzyme system consisting of three different isozymes: ALAD1-1, ALAD1-2, and ALAD2-2.Individuals dominantly expressing ALAD2, ALAD1-2 and ALAD2-2 have a higher susceptibility to Lead toxicity than those expressing the ALAD1-1 isozyme.The prevalence of the ALAD-2 allele is race specific and usually in the ranges of ranges from 0 to 20 percent [13].Therefore, the ALAD polymorphism affects and modifies Lead metabolism and delivery to target organs [18].To date, no study regarding ALAD enzyme activity and polymorphism distribution in Ugandan population.The present study, therefore, aimed at expounding the ALAD enzyme activity and the distribution of ALAD genotypes in relation to Lead exposure susceptibility in Uganda children.To our knowledge, this is the first study to address Lead exposure susceptibility, ALAD enzyme activity and polymorphism in Ugandan children.

Methods and methodology
This study was approved by Gulu University Research Ethics Committee No. (GUREC-048) dated 31/05/2019.Intentions of the study were first clearly explained in both English and the local language to the participants' parents/guardians prior to signing the informed consent forms.Venous blood samples (n = 198) equivalent to 5mls from a crosssection of children aged 6-60months residing in Katanga slum of Kampala city Uganda (00°18′49″N 32°34′52″E) coordinates were drawn into both hepatized and EDTA tubes by qualified nurses and technicians and transported on ice to Makerere University Biochemistry Department laboratory for analysis.Visibly malnourished children were excluded from this study.

Assay for blood Lead using atomic absorption spectrophotometer
Blood Lead levels were determined on an atomic absorption spectrophotometer (Agilent 2000 series) equipped with a graphite tube atomizer, a hollow-cathode lead lamp with a working current of 5mA, 283.3 nm spectral line and 0.5 nm bandwidth following a method described by [19].Five hundred microliter (500 µls) aliquots of blood samples were mixed with 1.2 ml of a solution containing 0.5% Triton X-100 and 1% (NH4)2HPO4.A total volume of 1.8 ml of deionized water was added to each sample in the tube and this was followed by the addition of 1.5 ml of 20% Trichloroacetic acid (TCA) prior to vortex mixing.The samples were centrifuged at 5000 rpm for 20 min and 10 µl of the supernatant from each was harvested and injected into the graphite tube.Lead standard concentrations ranged from 2 µg /dL to 50 µg /dL, the sample were analyzed in duplicate, and their mean value determined with occasional blanking with deionized/distilled water.The machine had a detection limit of 2μg/dL.

Colorimetric determination of hemoglobin levels by blood cyanmethemoglobin reaction method
Hemoglobin levels were determined following a cyanmethemoglobin reaction method described by [20].Aliquots equivalent to 100µl of samples in duplicates were made to a total volume of 1000µl with reaction solution containing 200mg of hexacyanoferrate III, 50mg of potassium cyanide, 140mg of potassium hydrogen phosphate and 1ml of Triton X-100 in a litre of distilled water.This was then incubated for 15 min at room temperature before reading at 540 nm, with the blank being the reaction reagent.Then 500 µl of standard hemoglobin standard (0.7mg/ml) was diluted with 500 µl of the same reagent, treated as above and readings taken.The Hb concentration in g/dl was calculated using formula; Where OD =optical density or absorbance at 540nm

Determination of hematocrit levels of the study blood samples
The hematocrit of the study blood samples was assayed following a method described by [21,22].Whole blood samples in heparinized tubes were forced into narrow diameter glass capillary tubes to two-third levels.The capillary tubes had self-sealing compound from one end.The capillaries together with the blood were loaded onto a microhematocrit centrifuge and ran at a relative centrifugal force of 14,000 × g for five minutes.Following centrifugation, hematocrit levels of each sample were measured within 10 minutes while the tubes were kept in a horizontal position to avoid merging of the layers.Hematocrit levels were estimated by calculating the ratio of the column of packed erythrocytes to the total length of the sample in the capillary tube.

Determination of delta-aminolevulinic acid dehydratase (ALAD) enzyme activity
The blood δ−ALAD enzyme activity in all the samples collected was measured following a method described by [23].
The ALAD enzyme activity of each sample in duplicate was determined by having 0.20mls incubated with 1.30mls of Triton X-100 reagent in disposable plastic tubes and there after adding 1 ml of buffered ALA substrate (0.01M).The buffered ALA substrate was prepared by dissolving 0.1676g of ALA-HCL in 100mls of phosphate-citrate buffer pH 6.65.The buffer was previously prepared by dissolving Na2HPO4 6.703g/dL (0.25M) and citric acid 5.25g/dL (0.25M).Aliquots equivalent to 1ml from each sample and blanks (distilled water) were added to tubes containing 1ml of TCA reagent.
To both test and blank aliquots, 1.0mls of the modified Ehrich's reagent previously prepared by dissolving 10g of pdimethylaminobenzaldehyde (DMBA), in 420mls of acetic acid and diluted to 1L with distilled water before storing at 4 0 C from which a working solution was made by mixing 50mls of DMBA-acetic acid with 8mls of 70% perchloric acid.Following the addition of the modified Ehrich's working reagent, the mixtures were allowed to stand for 13min for color to develop before measuring at 555 nm on a spectrophotometer.The corrected absorbance A = (Test absorbancethe blank absorbance) were used to calculate the activity of the enzyme.
Corrected Absorbance: A×1250 = units of ALAD enzyme activity, Hb concentration (g/dL) = OD sample × concentration of the standard (mg/dL) OD standard sample Hematocrit Where 12500 is the blood dilution factor.

Delta-aminolevulinic acid dehydratase (ALAD) Genotyping
Blood samples were analyzed following a method described by [24,25], the genomic DNA from blood samples was extracted using a Qiagen genomic DNA purification kit following the manufacturers instruction.The resultant DNA products were purified prior to polymerase chain rection (PCR) amplification.The PCR reaction mixture equivalent to 50-μL contained 1× buffer (10 mM Tris-HCl, pH 8.8; 50 mM KCl), 2 mM MgCl2, 0.2 mM dNTPs, 20 pmol each primers; (Forward, 5'-AGACAGACATTAGCTCAGTA-3', and reverse, 5'GGCAAAGACCACGTCCATTC-3') and 3U Taq DNA polymerase.The running conditions were; pre-denaturation at 94 °C for five min, followed by thirty-five cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, synthesis at 72 °C for 1 minutes and final extension at 72 °C for five min.The amplified products (916-bp region of genomic DNA) in volumes of 10 μL were digested overnight with MspI restriction enzyme (2.5 units) in a 20 μL reaction mixture containing 50 mM sodium chloride, 10 mM Tris-HCl, 10 mM magnesium chloride, 1mM dithiothreitol (pH 7.9) at 37 °C.The fragments were separated by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under UV illumination system.ALAD1-2 samples had both a 583-and a 512-bp fragment, whereas ALAD1-1 individuals had a single 583-bp fragment.

Data analysis;
Results were expressed as means, and the statistical significance evaluated by one way analysis of variance (ANOVA) using GraphPad Prism eight version.
Results Following genotyping the samples for ALAD alleles, the outcome is shown in the table 7.1 below with corresponding BLL, Hb levels, hematocrit and ALAD enzyme activities.The results indicate that ALAD1-1 allele is the most predominant with moderately high hemoglobin levels and seemingly normally functioning ALAD enzyme.The frequency of ALAD 2 allele is shown to be the least predominant as compared to ALAD1 allele.Comparing the hemoglobin levels across all the groups, it is apparent that ALD2 allele members have lower Hb levels.The results further indicate that members with genotype ALAD1-1 had their ALAD enzyme activity functioning moderately as compared to the rest.The results were statistically analyzed with Minitab 19 statistical software for correlation and significance and the results are shown in table 7.2 below.From the results, ALAD enzyme activity and hemoglobin levels strongly correlated with blood Lead levels across all the genotypes.

Discussion
Various factors including duration (time) of exposure [26] levels of environmental Lead pollutant in the area, nutritional status, age and the genetics accounts for one's Lead poisoning susceptibility [27,28].Even with similar environmental settings and confounding factors, variations in susceptibility to Lead poisoning among individuals exist [29,30].This study therefore aimed at expounding the relationship between genetic variations of proteins that code for ALAD enzyme and Lead susceptibility among individuals (children aged 6-60 months) living in the same geographical area (Katanga Uganda).Based on the available rich literature about the stoichiometric inhibitory effect of blood Lead ions on ALAD activity [31,32], we hypothesized that ALAD allele frequency distribution account for one's blood Lead levels.The extent of this ALAD enzyme inhibition is dependent on one's ALAD protein genetics [13].From the fact that this enzyme (ALAD) is polymorphic [33] with a G-to-C transversion at position 177 (db SNP ID: rs1800435) and two alleles (ALAD1 and ALAD2) with three isozymes; ALAD 1-1, ALAD 1-2, and ALAD 2-2, dominating ALAD allele accounts for his/her Lead toxicity susceptibility.Delta-aminolevulinic dehydratase enzyme polymorphism differ by race, and geographical location.Form the current study findings, we report significant correlations between ALAD genotype and Hb level, ALAD genotype and ALAD enzyme activity, and blood Lead levels (table 8.2).Blood Lead levels were elevated in carriers of ALAD 2-2 isozyme than those of both ALAD 1-2 and ALAD 1-1 isozyme carriers (table 8.1).The variations in Lead burden among these three groups observed in table 8.1 are attributed to the difference in the electronegativity of the amino-acids lysine and asparagine that code for these isozymes.From this study findings, we concur with reports of various previous studies from different regions that indicate low prevalence of ALAD 1-2 and ALA2-2 as compared to ALAD 1-1 genotype.Based on the study results in table 8.1, it is acceptable that having ALAD 1-2 and ALAD 2-2 alleles as the less predominant phenotypes delay Lead poisoning symptoms like ALAD enzyme inhibition [34,35], than individual who have ALAD 1-1 as the dominating allele.We statistically analyzed data groups (ALAD 1-1 vs ALAD 1-2 and 2-2 individuals) using one way analysis of variance (ANOVA).
Step-wise regression and multiple analyses of variance were used to assess the contribution effect of different ALAD genotypes towards blood Lead levels, ALAD enzyme activity and hemoglobin levels of the study participants (table 8.1).Compared to other isozymes, ALAD 1 -1 genotype which is encoded by the less electronegative protein (lysine) was the most dominant and because of this it binds less Pb ions hence more susceptibility to Lead poisoning.This is owed to the fact that Lead ions bind ALAD with high electronegative charge tightly hence reducing the amount of bioavailable Lead ions in circulation [36].The, unbound Lead ions circulating freely in the body systems end up affecting many vital organs.However, in times of oxidative and nutritional challenges, this tightly bond Lead is released back into circulation resulting manifestations like anemia, impaired intelligent quotient (IQ) etc.
These findings therefore, reveal that ALAD-2 allele variant modifies Lead kinetics by making it more available in circulation while lowering its uptake into cortical bones.
This study therefore, concludes that ALAD polymorphism is of great importance during the toxicokinetics of Lead poisoning during exposure and therefore recommend a further ALAD genotyping involving a bigger study population.

Ethics approval and consent to participate
This study was approved by Gulu University Research Ethics Committee No. (GUREC-048).

Consent for publication
Guardians and parents were clearly explained the study objectives to which they consented.

Introduction
• Your child is being asked to be in a research study on the Role of blood lead on anemia pathogenesis in children with malaria She /he was selected as a possible participant because she/he has malaria infection and is the resident of lead polluted area • We ask that you read this form and ask any questions that you may have before allowing your child to participate in this study.

Purpose of Study
• The purpose of the study is to investigate the interaction between blood lead levels and Plasmodium parasite infection during anemia pathogenesis in children under five years of age • Ultimately, this research may be published in journals

Description of the Study Procedures
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• The benefits of participation are [explain benefits of participation that will be gained by the participants and/or other.If a benefit is not likely to occur to each participant do not include.subject for this study, and that you have read and understood the information provided above.You will be given a signed and dated copy of this form to keep, along with any other printed materials deemed necessary by the study investigators.

Table 7 .1: The gene distribution of ALAD isozymes among the study participants.
Their corresponding levels of blood Lead, ALAD enzyme activity, hemoglobin and hematocrit.

values between different ALAD isozymes and blood Lead levels, ALAD en- zyme activity, hemoglobin levels and hematocrit volumes.
• [If there are no expected benefits, state as such.]Confidentiality • This study is anonymous.We will not be collecting or retaining any information about your child's identity.The decision to participate in this study is entirely up to you and your child.You are welcome to observe the interview if you wish.Your child may refuse to take part in the study at any time without affecting your relationship with the investigators of this study or Smith College or losing benefits to which you are otherwise entitled.Your child has the right not to answer any single question, as well as to withdraw completely from the interview at any point during the process; additionally, you have the right to request that the interviewer not use any of the interview material.Right to Ask Questions and Report Concerns • You have the right to ask questions about this research study and to have those questions answered by me before, during or after the research.If you have any further questions about the study, at any time feel free to contact me, Mukisa Ambrose] at amukisa@cns.mak.ac.ug] or by telephone at 0790222111.If you like, a summary of the results of the study will be sent to you.If you have any other concerns about your rights as a research participant that have not been answered by the investigators, you may contact Makerere University Institutional Review Board.• If you have any problems or concerns that occur as a result of your participation, you can report them to Dr. Kyambadde Joseph (supervisor) Consent • Your signature below indicates that you have decided to allow your child participate as a research