Possible influence of [delta]-aminolevulinic acid dehydratase polymorphism and susceptibility to renal toxicity of lead: a study of a Vietnamese population.
We examined six newly identified polymorphisms in the [delta]-aminolevulinic acid dehydratase dehydratase /de·hy·dra·tase/ (de-hi´drah-tas) a common name for a hydro-lyase.
n. (ALAD ALAD
d-aminolevulinic acid dehydratase. ) single-nucleotide polymorphisms (SNPs) to determine if these SNPs could modify the relationship between blood lead (PbB) and some renal parameters. This is a cross-sectional study cross-sectional study
See synchronic study.
n the scientific method for the analysis of data gathered from two or more samples at one point in time. of 276 lead-exposed workers in Vietnam. All workers were measured for PbB, urinary retinol-binding protein (URBP), urinary [c.sub.1]-microglobulin (U[alpha]1m), urinary [[beta].sub.2]-microglobulin (U[beta]2m), urinary N-acetytl-[beta]-D-glucosaminidase (NAG 1. NAG - Numerical Algorithms Group.
2. NAG - The Linux Network Administrators' Guide. ), urinary aminolevulinic acid aminolevulinic acid /ami·no·lev·u·lin·ic ac·id/ (ALA) (-lev?u-lin´ik) d-aminolevulinic acid; an intermediate in the synthesis of heme; blood and urinary levels are increased in lead poisoning, and urinary levels are increased in some (ALAU ALAU Delta-Amino-Levulinic Acid Dehydratase in Urine (lead poisoning indicator) ), serum [[alpha].sub.1]-microglobulin (S[alpha]m), serum [[beta].sub.2]-microglobulin (S[beta]2m), and urinary albumin (Ualb). The six SNPs were Msp and Rsa in exon Exon
In split genes, a portion that is included in the ribonucleic acid (RNA) transcript of a gene and survives processing of the RNA in the cell nucleus to become part of a spliced messenger RNA (mRNA) or structural RNA in the cell cytoplasm. 4, Rsa39488 in exon 5, HpyIV and HpyCH4 in intron Intron
In split genes, a portion that is included in ribonucleic acid (RNA) transcripts but is removed from within a transcript during RNA processing and is rapidly degraded. 6, and Sau3A in intron 12. Analysis of covariance Covariance
A measure of the degree to which returns on two risky assets move in tandem. A positive covariance means that asset returns move together. A negative covariance means returns vary inversely. (ANCOVA ANCOVA Analysis of Covariance ) with interaction of PbB x SNPs were applied to examine modifying effect of the SNPs on the association of renal parameters and PbB, adjusting for potential confounders of age, gender, body mass index, and exposure duration. HpyCH4 was found to be associated with certain renal parameters. For HpyCH4 1-1, an increase of I [micro]g/dL PbB caused an increase of 1.042 mg/g creatinine creatinine /cre·at·i·nine/ (kre-at´i-nin) an anhydride of creatine, the end product of phosphocreatine metabolism; measurements of its rate of urinary excretion are used as diagnostic indicators of kidney function and muscle mass. (Cr) U[alpha]1m, 1.069 mg/g Cr U[beta]2m, 1.038 mg/g Cr URBP, and 1.033 mg/g Cr Ualb, whereas in HpyCH4 1-2, an increase of 1 [micro]g/dL PbB resulted in an increase of only 1.009 mg/g Cr U[alpha]1m, 1.012 mg/g Cr U[beta]2m, 1.009 mg/g Cr URBP, and 1.007 mg/g Cr Ualb. HpyCH4 SNP SNP Scottish National Party
Noun 1. SNP - (genetics) genetic variation in a DNA sequence that occurs when a single nucleotide in a genome is altered; SNPs are usually considered to be point mutations that have been evolutionarily appeared to modify the lead toxicity to kidney with wild-type allele allele (əlēl`): see genetics.
Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. being more susceptible than variants. The mechanism for this effect is not clear. Further studies are needed to confirm this observation. Key words: [delta]-aminolevulinic acid dehydratase (ALAD), HpyCH4, intron, lead, SNP (single-nucleotide polymorphism polymorphism, of minerals, property of crystallizing in two or more distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing as calcite or aragonite. Titanium dioxide is trimorphous; its three forms are brookite, anatase (or octahedrite), and rutile. ), urinary albumin (Ualb), urinary retinol-binding protein (URBP), urinary [[alpha].sub.1]-microglobulin (U[alpha]m), urinary [[beta].sub.2]-microglobulin (U[beta]2m). Environ Health Perspect 113:1313-1317 (2005). doi:10.1289/ehp.7904 available via http://dx.doi.org/[Online 1 June 2005]
The first and most common [delta]-aminolevulinic acid dehydratase (A/AD) polymorphism studied was the Msp single-nucleotide polymorphism (SNP) in exon 4. Since it was first reported (Battistuzzi et al. 1981), many reports (e.g., Onalaja and Claudio 2000) have been published on its association with inorganic lead (henceforth referred to as lead). Epidemiologic studies have tried to determine if the Msp polymorphism is important in human susceptibility to lead regarding various lead-targeted systems (Alexander et al. 1998; Bergdahl et al. 1997; Hu et al. 2001; Schwartz et al. 1995, 2000).
In recent years, reports have been published on the association between the Msp SNP and renal effects of lead exposure. Smith et al. (1995) suggested that the Msp-2 variant is a more susceptible allele because Msp-2 carriers had higher concentrations of blood uric acid uric acid (yr`ĭk), white, odorless, tasteless crystalline substance formed as a result of purine degradation in man, other primates, dalmatians, birds, snakes, and lizards. and blood urea nitrogen blood urea nitrogen
n. Abbr. BUN
Nitrogen in the form of urea in the blood or serum, used as a indicator of kidney function.
Blood urea nitrogen (BUN) (BUN). The differences achieved only borderline significance after adjusting for potential confounders. Bergdahl et al. (1997) found that serum creatinine is higher for Msp)-2 carriers than for Msp 1-1 homozygotes in a sample of 89 lead workers. A linear relationship was observed between patella patella (pətĕl`ə): see kneecap. lead concentration, using X-ray fluorescence X-ray fluorescence (XRF) is the emission of characteristic "secondary" (or fluorescent) X-rays from a material that has been excited by bombarding with high-energy X-rays or gamma rays. method, and serum uric acid when patellar patellar
of or pertaining to the patella.
a cartilaginous process borne on the medial side of the patella of horses and cattle. lead passes 15 [micro]g/g bone mineral for Msp-2 carriers, but the similar linear relation can be seen only when patella lead was > 101 [micro]g/g bone mineral for the Msp 1-1 group (Wu et al. 2003). Wu et al. (2003) postulated pos·tu·late
tr.v. pos·tu·lat·ed, pos·tu·lat·ing, pos·tu·lates
1. To make claim for; demand.
2. To assume or assert the truth, reality, or necessity of, especially as a basis of an argument.
3. that lead toxicity became apparent at a lower exposure level for Msp-2 carriers than for Msp 1-1 persons. These studies suggested that Msp-2 carriers were more susceptible to lead effects than were Msp 1-1 homozygotes. However, the reports were not consistent. Some studies had reported that the Msp-2 variant was associated with higher creatinine clearance creatinine clearance
The volume of serum or plasma that would be cleared of creatinine by one minute's excretion of urine.
creatinine clearance as well as lower mean serum creatinine and BUN (Weaver et al. 2003).
Renal parameters such as creatinine clearance, BUN, and serum creatinine are insensitive to renal function In medicine (nephrology) renal function is an indication of the state of the kidney and its role in physiology. Indirect markers
Most doctors use the plasma concentrations of creatinine, urea, and electrolytes to determine renal function. changes. As much as 50-70% of kidney nephrons must be damaged before variation can be detected (Goyer 1989). The consensus may not be so clear, however, regarding the effects of lead on the renal system because no good longitudinal studies longitudinal studies,
n.pl the epidemiologic studies that record data from a respresentative sample at repeated intervals over an extended span of time rather than at a single or limited number over a short period. have been conducted to establish the predictive value pre·dic·tive value
The likelihood that a positive test result indicates disease or that a negative test result excludes disease.
a measure used by clinicians to interpret diagnostic test results. of early biologic exposure markers in lead-exposed workers. Roels et al. (1994) studied 76 male lead smelter workers and 68 controls matched for age, gender, socioeconomic state, residence, and work shift characteristics. Although the tibia tibia: see leg. bone lead, blood lead (PbB), and urinary lead levels of the exposure group were significantly higher than those of the control group, no significant differences were observed in either common and stimulated creatinine clearance. More recently, however, Weaver et al. (2003) reported that higher lead levels were associated with lower BUN and serum creatinine levels and higher calculated creatinine clearance among those with the ALAD 1-2 genotype genotype (jēn`ətīp'): see genetics.
Genetic makeup of an organism. The genotype determines the hereditary potentials and limitations of an individual. .
Renal parameters such as low-molecular-weight proteins [e.g., retinol-binding protein, [[alpha].sub.1]-microglobulin, [[beta].sub.2]-microglobulin, N-acetyl-[beta]-D-glucosaminidase (NAG)] representing proximal tubule The proximal tubule is the portion of the duct system of the nephron leading from Bowman's capsule to the loop of Henle. Structure and appearance
The most distinctive characteristic of the proximal tubule is its brush border (or "striated border"). injury are considered good alternatives because early lead-induced nephropathy nephropathy /ne·phrop·a·thy/ (ne-frop´ah-the) disease of the kidneys.nephropath´ic
analgesic nephropathy usually involves damage of proximal tubule cell (Nolan and Shaikh 1992). To date, several studies have shown that these renal parameters have good correlation with lead exposure indices. NAG has been found to be the only marker elevated in early nephropathy in five studies reviewed by Bernard and Lauwerys (1989). One study in Japan (Endo et al. 1993) claimed that urinary [[alpha].sub.1]-microglobulin (U[[alpha].sub.1]m) can be a useful indicator of renal impairment. A study of 128 lead workers (Chia et al. 1995) reported that U[[alpha].sub.1]m appears to be the most sensitive parameter compared with urinary [[beta].sub.2]-microglobulin (U[[beta].sub.2]m) and urinary retinol-binding protein (URBP). Another study of environmental lead exposure in children showed that URBP is increased by lead with good dose-effect and dose-response relations with PbB (Bernard et al. 1995).
To date, 111 SNPs have been reported in the National Center for Biotechnology Information The National Center for Biotechnology Information (NCBI) is part of the United States National Library of Medicine (NLM), a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988. website (NCBI NCBI National Center for Biotechnology Information (NIH)
NCBI National Coalition Building Institute
NCBI National Council for the Blind of Ireland (Dublin, Ireland) 2005). But when we assessed the website in June 2004, 46 SNPs were reported. What are the relationships of these 46 SNPs with the renal effect of lead exposure? We used the Helix Tree software (Golden Helix, Inc., Bozeman, MT, USA; http://www.goldenhelix.com/pharmhelix treefeatures.html) to test for linkage disequilibrium linkage disequilibrium
The nonrandom association between two or more alleles such that certain combinations of alleles are more likely to occur together on a chromosome than other combinations of alleles. among the 46 SNPs; 6 SNPs were shown not to be in linkage disequilibrium. In this study we examined these 6 SNPs (Msp and Rsa in exon 4, Rsa39488 in exon 5, HpyIVand HpyCH4 in intron 6, Sau3A in intron 12) and examined their association with certain renal functions among a group of lead-exposed workers in Vietnam.
Materials and Methods
Study population. The study population consisted of 323 workers from a battery factory in Hal Phong City, Vietnam. All workers from the production line as well as in the division of management and quality control were recruited into this study. Of these 323 workers, 246 were occupationally exposed to lead, and the remaining 77 were not directly exposed to lead. Although these 77 workers were not directly exposed to lead, many had a previous history of exposure. Some were still exposed, albeit less than the 246 group. Therefore, these workers were also included in the study. Signed consent was obtained for each worker before blood and urine samples were taken for subsequent analysis. The workers also filled out a questionnaire (in Vietnamese) with the help of a Vietnamese interviewer. After completing the questionnaire, a spot urine sample and 10 mL of blood were collected from each worker during the medical examination. Twenty of the exposed lead workers were not present during the study period, and 17 did not want to participate. Of the 77 workers who were not directly exposed to lead, 10 were unwilling to give their urine and blood for analysis. Because the study was strictly on a voluntary basis, the workers' decisions in not giving urine and/or blood were respected. Hence, the 47 workers were excluded in the study, giving a response rate of 88.2% (276 of 323).
Questionnaire. Information gathered included age, years of education, detailed occupational history, and current and previous smoking habits. Alcohol intake was also documented carefully, as it could confound con·found
tr.v. con·found·ed, con·found·ing, con·founds
1. To cause to become confused or perplexed. See Synonyms at puzzle.
2. the findings. Actual amount of alcohol consumption per day (i.e., types of drink consumed and the amount estimated by number of bottles consumed) and number of years of drinking were noted. Six workers gave a history of diabetes mellitus diabetes mellitus
Disorder of insufficient production of or reduced sensitivity to insulin. Insulin, synthesized in the islets of Langerhans (see Langerhans, islets of), is necessary to metabolize glucose. In diabetes, blood sugar levels increase (hyperglycemia). and/or hypertension. These workers were excluded from the study because diabetes mellitus and hypertension are known to affect renal functions.
Laboratory analysis. We obtained blood samples by venipuncture venipuncture /veni·punc·ture/ (ven?i-pungk´chur) surgical puncture of a vein.
ve·ni·punc·ture or ve·ne·punc·ture
n. with lead-flee disposable syringes and stored the samples in heparinized lead-free polypropylene tubes. PbB was analyzed using atomic absorption spectrophotometry spectrophotometry
Branch of spectroscopy dealing with measurement of radiant energy transmitted or reflected by a body as a function of wavelength. The measurement is usually compared to that transmitted or reflected by a system that serves as a standard. (Varian Spectra AA-30; SiberHegner Pte Ltd PTE LTD Private Limited ., Victoria, Australia) with a graphite furnace. External quality control was performed yearly under the National External Quality Assurance Scheme (England) and Inter-laboratory Comparison Programme (Canada). We obtained spot urine samples, and those used for analysis of ALAU, URBP, U[alpha]1m, U[beta]2m, urinary albumin (Ualb), and NAG were buffered (pH 7.2). Serum and urine samples were stored at -30[degrees]C until analysis. We analyzed all samples within 1 month of arrival in Singapore at our laboratory and adjusted all urine parameters for variability in urine flow using urinary creatinine that was expressed per gram of creatinine. Urine creatinine concentrations were determined using standard laboratory techniques Laboratory techniques are the sum of procedures used on natural sciences such as chemistry, biology, physics in order to conduct an experiment, all of them follow scientific method; while some of them involves the use of complex laboratory equipment from laboratory glassware to . Ualb, ALAU, URBP, U [alpha]1m, U[beta]2m, and NAG were measured by enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA)
A diagnostic blood test used to screen patients for AIDS or other viruses. using commercially available polyclonal antibodies or test kits (Roche Diagnostic Corp., Indianapolis, IN, USA). The details of these tests have been reported previously (Green et al. 2004). NAG was determined using Noto's method (Chia et al. 1995)
Identification of ALAD polymorphisms. Six polymorphisms located on the ALAD gene were selected for analyses in our study. They are MspI and RsaI polymorphisms, which span a 248-bp region of the genomic ALAD sequence, as well as Rsa39488 (232 bp), HpyIV(234 bp), HpyCH4 (213 bp), and Sau3AI (282 bp) polymorphisms. The products of interest were determined through amplification by polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) ), using primers designed with Primer3 (Whitehead Institute Founded in 1982, the Whitehead Institute for Biomedical Research is a non-profit research and teaching institution located in Cambridge, Massachusetts. The Whitehead Institute was founded as a fiscally independent entity from Massachusetts Institute of Technology, and its members for Biomedical Research Biomedical research (or experimental medicine), in general simply known as medical research, is the basic research or applied research conducted to aid the body of knowledge in the field of medicine. , Cambridge, MA, USA; bttp://frodo.wi.mit. edu/cgi-bin/primer3/primer3_www.cgi). The amplification cycles were performed on the PTC-100 MJ Thermal Cycler The Thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus used for PCR. The device has a thermal block with holes where tubes with the PCR reaction mixtures can be inserted. (PerkinElmer, Inc., Boston, MA, USA) and optimized to ensure specific amplification of the products of interest. Each PCR reaction was performed in a final volume of 25 [micro]L, which included 10 ng of pure genomic DNA genomic DNA
The full complement of DNA contained in the genome of a cell or organism. , 1.0 [micro]M each primer, 200 [micro]M each dNTP, 1x Taq buffer, 1.5 mM Mg[Cl.sub.2], and 1.0 U of Taq DNA polymerase DNA polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes catalyzing the template-directed incorporation of deoxyribonucleotides into a DNA chain, particularly one using a DNA template. (all from Promega Corp., Madison, WI, USA), and topped up to the required volume with sterile distilled water Noun 1. distilled water - water that has been purified by distillation
H2O, water - binary compound that occurs at room temperature as a clear colorless odorless tasteless liquid; freezes into ice below 0 degrees centigrade and boils above 100 degrees centigrade; . We used the restriction fragment length polymorphism restriction fragment length polymorphism
n. Abbr. RFLP
Intraspecies variations in the length of DNA fragments generated by the action of restriction enzymes and caused by mutations that alter the sites at which these enzymes act, changing method to locate the site of mutation; the fragments were determined on a 2.0% agarose agarose
more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments. gel (Seakem agarose; Cambrex North Brunswick Inc., North Brunswick, NJ, USA) and visualized on the Biorad Gel Doc 2000 system(BioRad Laboratories, Hercules, CA, USA).
Statistical analysis. We determined that 28 people did not have any genotype measurements and had to exclude them from the analysis, leaving a sample of 248 workers. The data set of these 248 workers was used in all subsequent analyses. For some of the biologic samples, we were not able to analyze all the parameters because these samples ran out before we could complete the test.
The distributions of variables were checked individually with histogram histogram
or bar graph
Graph using vertical or horizontal bars whose lengths indicate quantities. Along with the pie chart, the histogram is the most common format for representing statistical data. and one-sample Kolmogorov-Smimov (K-S K-S Kolmogorov-Smirnov (statistical test) ) test for normality. Necessary transformation was undertaken to achieve normal distribution for all important variables. In the end, ALAU, U[alpha]1m, U[beta]2m, Ualb, URBP, serum [[alpha].sub.1]-microglobulin (S[alpha]1m), and serum [[beta].sub.2]-microglobulin (S[beta]2m) went through logarithmic logarithmic
pertaining to logarithm.
when the logs of two variables plotted against each other create a straight line. transformation. NAG went through square root transformation. The transformed variables all follow normal distribution with an insignificant p-value from the one-sample K-S test.
Statistical analysis addressed two issues: a) Is there any difference in the mean PbB concentrations and renal parameters between genotypes of each SNP after adjusting for known confounders? b) Within each single SNP, is the effect of PbB concentration on the renal parameters similar across genotype subgroups? Analysis of covariance (ANCOVA; Kumer et al. 2003) was the main statistical technique used. We initially examined scatter plots of each renal parameter (y-axis transformed value) and PbB concentration (x-axis) to identify linearity and extreme points.
We addressed the first issue using ANCOVA to adjust for age, exposure duration, body mass index (BMI BMI body mass index.
body mass index
Body mass index (BMI)
A measurement that has replaced weight as the preferred determinant of obesity. ), PbB (where appropriate), and gender. For the second issue, an interaction term of PbB x SNP was included while adjusting for the known confounders. Multiply linear regression Linear regression
A statistical technique for fitting a straight line to a set of data points. equation models were used in the generation of regressions lines for Figures 1-4. All data are processed by SPSS A statistical package from SPSS, Inc., Chicago (www.spss.com) that runs on PCs, most mainframes and minis and is used extensively in marketing research. It provides over 50 statistical processes, including regression analysis, correlation and analysis of variance. (version 12.0; SPSS Inc., Chicago, IL, USA). Significance level was set as p < 0.05 (two-sided).
Of the 248 workers, 184 (74.2%) were male and 64 (25.8%) were female, with a mean age of 39 (range, 204;6) years. The range of exposure duration was 1-41 years. Demographic features by SNP are summarized in Table 1. The allelic al·lele
One member of a pair or series of genes that occupy a specific position on a specific chromosome.
[German Allel, short for Allelomorph, allelomorph, from English and genotypic genotypic
emanating from or pertaining to genotype.
selection of breeding stock on the basis of known inherited characteristics. frequencies are summarized in Table 2. For Msp, Sau3A, HpyIV, and HpyCH4, the wild-type allele is more predominant, whereas for Rsa and Rsa39488, the variant allele is morn prevalent. The basic characteristics of the measurement variables are shown in Table 3. Table 4 shows the comparison of mean renal parameters by SNPs. The HpyIV variant group had higher mean ALAU and Ualb levels than the group with the wild-type genotype (1.15 vs. 0.95 mg/g Cr and 7.75 vs. 7.37 mg/g Cr, respectively). The wild-type genotype group for the Sau3A marker (1-1) had lower ALAU levels compared with those of the variant group (0.93 vs. 1.08 mg/g Cr) but higher NAG concentrations (2.93 vs. 2.53 U/g Cr). Only Rsa39488 subgroups had significant differences in PbB, with means of ALAD 1-1, ALAD 1-2, and ALAD 2-2 being 21.87 [micro]g/dL, 20.43 [micro]g/dL, and 25.54 [micro]g/dL, respectively. The group with the Rsa39488 variant genotype, 2-2, had much higher PbB levels than the 1-1 and 1-2 genotype groups (Table 4).
Multiple linear regression equation models were constructed for each of the six SNPs. Of these six SNPs, only HpyCH4 showed significant association with URBP, U[alpha]1m, and U[beta]2m in the multiple regression Multiple regression
The estimated relationship between a dependent variable and more than one explanatory variable. analysis. Table 5 shows the relationship between different renal parameters and the HpyCH4 SNP. PbB concentrations significantly affect URBP, U[alpha]1m, U[beta]2m, and Ualb even after adjusting for age, gender, BMI, and exposure duration. PbB is an important predictor for these renal indices. There are significant interactions between the HpyCH4 marker and PbB concentration (Table 5). The associations of PbB concentrations and renal parameters are presented in Figures 1-4.
[FIGURES 1-4 OMITTED]
The increment in renal parameters corresponding to unit increase in PbB concentration is higher for HpyCH4 1-1 homozygotes than for HpyCH4 1-2 workers. For U[alpha]1m, each increase of 1 [alpha]g/dL PbB corresponds to an increase of 0.016 log U [alpha]1m (antilog an·ti·log
Noun 1. antilog - the number of which a given number is the logarithm
antilogarithm 1.042 mg/g Cr) for HpyCH4 1-1 workers, whereas for HpyCH4 i-2 workers the increase is 0.004 (anti-log 1.009 mg/g Cr). The same trend is shown in the relations of U[beta]2m, URBP, and Ualb to PbB concentrations. The contrast between HpyCH4 1-1 and HpyCH4 1-2 is 1.069 versus 1.012 mg/g Cr, 1.038 versus 1.009 mg/g Cr, and 1.033 versus 1.007 mg/g Cr for U[beta]2m, URBP, and Ualb, respectively.
For ALAD SNPs, this study is the first to address more than one SNP. In addition to Ma[p in exon 4, we also examined Rsa SNP in exon 4, Rsa39488 in exon 5, HpyCH4 and HpyIV in intron 6, and Sau3A in intron 12. It has been shown by linkage analyses that these six SNPs are not in linkage disequilibrium.
We did not find any correlation of lead with S[alpha]1m or S[beta]2m, which is consistent with other studies (Cardenas et al. 1993; Chia et al. 1994; Endo et al. 1993). In our study, Msp SNP in exon 4 constitutes 95.9% of ALAD1 and 4.1% of ALAD2 alleles. This pattern is similar to those in most studies conducted on Asian populations (Hsieh et al. 2000; Shen Shen, in the Bible, place, perhaps close to Bethel, near which Samuel set up the stone Ebenezer. et al. 2001). The allele composition of the other five SNPs had not been reported. To our knowledge, this is the first report of genetic distribution of the ALAD polymorphism in a Vietnamese population.
Although we determined some positive findings with Rsa, Rsa39488, HpyIV, and Sau3A, our findings were not consistent. Therefore, these SNPs are not discussed further; we concentrate our discussion on HpyCH4 and its association with renal function. HpyCH4 is a SNP that involves a G/C G/C Gas-to-Cloth Ratio transversion trans·ver·sion
Eruption of a tooth in a position normally occupied by another.
n eruption of a tooth in the wrong position in gene position 12,916 in intron 6. This SNP was first reported by Olson (2000). Allele frequency allele frequency
The percentage of a population of a species that carries a particular allele on a given chromosome locus. in his sample (North American North American
named after North America.
North American blastomycosis
see North American blastomycosis.
North American cattle tick
see boophilusannulatus. population) was 0.818 for HpyCH4-1 and 0.182 for HpyCH4-2, and genotype frequency In population genetics, the genotype frequency is the frequency or proportion (i.e. 0 < f < 1) of genotypes in a population.
It may be denoted thus:
Compare allele frequency. is 0.727 for HpyCH4 1-1, O. 182 for HpyCH4 1-2, and 0.091 for HpyCH4 2-2. Nakamura (2002) reported the frequencies of HpyCH4-1 to be 0.808 and of HpyCH4-2 to be 0.191 in a sample of 734 Japanese. In our study, HpyCH4-1 has a frequency of 0.942, and HpyCH4-2 has a frequency of 0.058. HpyCH4 1-1 homozygotes accounted for 88.4% (19l), HpyCH4 1-2 heterozygotes accounted for 11.6% (25). Variant HpyCH4 2-2 homozygotes were not detected in this study population. Our study frequencies of the HpyCH4 allele were fairly similar to those reported by Nakamura (2002), whose sample was also drawn from an Asian population. However, the proportion of HpyCH4-1 in our study was relatively higher.
Several studies have shown that URBP (Bernard et al. 1995), U[alpha]1m (Chia et al. 1995; Endo et al. 1993), U[beta]2m (Nolan and Shaikh 1992), and urinary NAG (Bernard and Lauwerys 1989) are good indicators of early renal effects due to lead exposure. A study of 128 lead workers in Singapore (Chia et al. 1995) showed that U[alpha]1m appears to be the most sensitive parameter compared with U[beta]2m and URBP.
Our study found that a newly identified ALAD polymorphism, HpyCH4 in intron 6, was able to modify the association of PbB concentrations with certain renal parameters. For HpyCH4 1-1 homozygotes, 1 [micro]g/dL PbB caused an increase of 1.042 mg/g Cr U[alpha]1m, 1.069 mg/g Cr Ub2m, 1.038 mg/g Cr URBP, and 1.033 mg/g Cr Ualb, whereas in HpyCH4 1-2 heterozygotes, an increase of 1 [micro]g/dL PbB resulted in an increase of only 1.009 mg/g Cr U[alpha]1m, 1.012 mg/g Cr U[beta]2m, 1.009 mg/g Cr URBP, and 1.007 mg/g Cr Ualb. These findings suggest that the HpyCH4 1-2 variant could be more resistant to the effects of lead toxicity on some renal functions than HpyCH4 1-1.
We showed that in the relationship between PbB and some renal parameters (Figures 1-4), two regression lines (HpyCH4 1-1 and HpyCH4 1-2) intersect around 25 [micro]g/dL PbB. This point highlights the importance of stratifying the data by PbB when we study the modification effects of ALAD polymorphism on associations between lead exposure and renal function. Otherwise, problems would arise and cause bias in the findings. In our study, for workers with PbB < 25 [micro]g/dL, HpyCH4 2-2 workers have higher renal function values than HpyCH4 1-1 workers. The opposite is true for workers with PbB > 25 [micro]g/dL. Thus, one may conclude that ALAD2 is more susceptible when the samples have low lead exposure. Conversely, one might find that ALAD1 is more susceptible when the samples have higher exposure (i.e., PbB > 25 [micro]g/dL). Our findings may explain, to some degree, why there were contradicting reports on the association between PbB concentrations and ALAD alleles in some studies. Wetmur et al. (1991) found a significant overrepresentation of Msp-2 isozymes among individuals with PbB in excess of 30 [micro]g/dL. Schwartz et al. (1995) reported that the overrepresentation of Msp-2 allele can be present only when the PbB was > 40 [micro]g/dL. Smith et al. (1995) failed to show any association of Msp-2 to PbB and he ascribed the nonassociation with the low lead exposure of the study population (mean PbB, 7.78 [micro]g/dL).
For the May polymorphism, the differences noted in relation to the susceptibility of effects of lead have been attributed to the differential binding abilities of the ALAD isozymes. However, the HpyCH4 SNP, located in the intron region of the ALAD gene, technically does not have any protein-coding functions involved. George and David (1998) observed that some DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. behaves as an exon when expressed by one pathway but as an intron when expressed by another pathway. Both pathways can operate simultaneously, resulting in greater protein product variety. Perhaps the ALAD enzyme encoded by HpyCH4 1-1 and HpyCH4 1-2 could have been produced through one such pathway (George and David 1998). Regulatory functions of introns may involve controlling gene activity in different developmental stages or responding to immediate biologic needs by controlling local gene expressions. This function of introns could occur if exons code for a domain, a polypeptide polypeptide: see peptide. unit that has a discrete function such as binding to a membrane, the catalytic site of an enzyme, or a structural unit of a protein (Jerry 2001). However, few studies have reported that polymorphisms located in introns can be associated with lead toxicity. One study that examined Map polymorphism in intron 2 reported that variant workers were associated with decreased bone lead but not PbB (Kamel et al. 2003). Recent work has demonstrated that intronic mutations can have functional consequences in some genes such as p53 (Lehman et al. 2000). Similarly, intronic mutations may also have functional consequences in the ALAD genes.
Some limitations are inherent in this study. It is possible that individuals with certain ALAD polymorphisms may be more susceptible to the effects of lead toxicity on the renal system and thus would have been excluded from the workforce even before this study was conducted (healthy worker effect). But these susceptible workers will need to have obvious clinical renal diseases to be removed from the workforce. This is first time that factory workers in Vietnam were tested with these renal tests. Therefore, it is highly unlikely that we are dealing with a healthy worker effect. In Viemam, most factories are state owned, as is this factory. Workers work in a factory all their lives (as can be seen by the long exposure history in Table 1). Schwartz et al. (1995) reported differences in the Msp ALAD genotype frequencies among workers in three lead factories with different lead exposures. They postulated that workers with the more susceptible ALAD genotype may moved to a lower lead-exposed job. Conversely, workers with the protective ALAD genotype may continue working because they have fewer symptoms even in higher exposure areas. We are unable to study this possible "population stratification" bias because our workers were rotated through the different departments/sections in the factory depending on the job schedule and manpower requirement.
We did not measure the body burden of lead and thus could not examine the effects of the ALAD SNPs in relation to lead accumulation in the body. We did use exposure duration as a surrogate, and this factor has been adjusted for in our analysis. In spite of factoring in the duration of exposure to lead, workers with HpyCH4 1-2 genotype had significantly better renal (certain) parameters than workers with the HpyCH4 1-1 genotype. The sample size of our study is also not large. There were only 25 workers with HpyCH4 1-2 compared with 191 workers with HpyCH4 1-1. Cadmium cadmium (kăd`mēəm) [from cadmia, Lat. for calamine, with which cadmium is found associated], metallic chemical element; symbol Cd; at. no. 48; at. wt. 112.41; m.p. 321°C;; b.p. 765°C;; sp. gr. 8. is also known to affect some of the renal parameters that were measured in this study (Chia et al. 1989). It has been reported that urinary cadmium of 4 [micro]g/g Cr is the critical value for cadmium to have a significant effect on U[alpha]1m, U[beta]2m, URBP, and NAG (Rods et al. 1993). There was no measurement of cadmium levels for all the workers. However, we randomly selected a sample of 25 workers and determined their urine cadmium level; the mean was 0.57 [micro]g/g Cr with a range of 0.34-0.98 [micro]g/g Cr. These values were far below the reported urinary cadmium of 4 [micro]g/g Cr that would affect some of the studied renal parameters.
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Sin Eng Chia, (1) Huijun Zhou, (1) Mei Theng Tham, (1) Eric Yap, (1) Nguyen-Viet Dong, (2) NguyenThi Hong Tu, (3) and Kee Seng Chia (1)
(1) Department of Community, Occupational and Family Medicine, National University of Singapore The National University of Singapore (Abbreviation: NUS) is Singapore's oldest university. It is the largest university in the country in terms of student enrollment and curriculum offered. , Singapore, Republic of Singapore Noun 1. Republic of Singapore - a country in southeastern Asia on the island of Singapore; achieved independence from Malaysia in 1965
ASEAN, Association of Southeast Asian Nations - an association of nations dedicated to economic and political ; (2) Center of Occupational Health and Environment, Ministry of Industry, Hanoi, Vietnam; (3) General Department of Preventive Medicine preventive medicine, branch of medicine dealing with the prevention of disease and the maintenance of good health practices. Until recently preventive medicine was largely the domain of the U.S. and Control of HIV/AIDS HIV/AIDS Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome Control, Ministry of Health, Hanoi, Vietnam
Address correspondence to S-E S-E Spheno Ethmoidectomy . Chia, Department of Community, Occupational and Family Medicine (MD3), Faculty of Medicine, National University of Singapore, 16 Medical Dr., Singapore 117597, Republic of Singapore. Telephone: 65-68744970. Fax: 65-67791489. E-maih firstname.lastname@example.org
We express our appreciation to T.B. Choo, G. Wei, and C.Y. Huak for their generous assistance in the statistical analysis. We thank the management and workers for their participation.
This study was supported by research grant from the Agency for Science, Technology and Research The Agency for Science, Technology and Research (Abbreviation: A*STAR; Chinese: 新加坡科学技术研究局) is a statutory board in Singapore. , Biomedical Research Council The Biomedical Research Council (Abbreviation: BMRC; Simplified Chinese: 生物医药研究理事会) is a research council in Singapore, established in October 2000. , Singapore (grant 01/1/21/19/186).
The authors declare they have no competing financial interests.
Received 3 January 2005; accepted 1 June 2005.
Table 1. Demographic characteristics of the study population by SNPs. Gender Age (years) SNP Genotype Male [n(%)] Female [n(%)] n(%) Msp 1-1 168 (92.8) 58 (92.1) 226 (92.6) 1-2/2-2 13 (7.2) 5 (7.9) 18 7.4) Rsa 1-1 40 (22.1) 16 (26.2) 56 (23.1) 1-2 86 (47.5) 28 (45.9) 114 (47.1) 2-2 55 (30.4) 17 (27.9) 72 (29.8) Rsa39488 1-1 41 (23.0) 15 (23.8) 56 (23.2) 1-2 81 (45.5) 32 (50.8) 113 (46.9) 2-2 56 (31.5) 16 (25.4) 72 (29.9) HpyCH4 1-1 136 (87.2) 55 (91.7) 191 (88.4) 1-2 20 (12.8) 5 (8.3) 25 (11.6) HpyIV 1-1 131 (74.9) 42 (67.7) 173 (73.0) 1-2/2-2 44 (25.1) 20 (32.3) 64 (27.0) Sau3A 1-1 106 (66.7) 31 (51.7) 137 (62.6) 1-2/2-2 53 (33.3) 29 (48.3) 82 (37.4) Age (years) Exposure (years) SNP Mean [+ or -] SD n Mean [+ or -] SD Range Msp 39.5 [+ or -] 10.6 214 15.4 [+ or -] 10.2 1-41 36.4 [+ or -] 9.8 18 10.8 [+ or -] 9.1 1-27 Rsa 39.8 [+ or -] 11.0 54 15.8 [+ or -] 11.2 1-41 38.4 [+ or -] 10.3 107 14.3 [+ or -] 9.9 1-39 39.7 [+ or -] 10.7 69 15.0 [+ or -] 9.9 1-36 Rsa39488 40.0 [+ or -] 11.0 55 16.0 [+ or -] 11.3 1-41 39.3 [+ or -] 10.2 105 14.9 [+ or -] 10.0 1-39 39.0 [+ or -] 10.7 69 14.5 [+ or -] 9.5 1-36 HpyCH4 39.2 [+ or -] 10.4 184 15.5 [+ or -] 10.2 1-41 40.9 [+ or -] 10.7 23 13.9 [+ or -] 9.3 1-30 HpyIV 39.8 [+ or -] 10.4 163 15.3 [+ or -] 10.2 1-41 38.1 [+ or -] 10.9 62 14.4 [+ or -] 10.3 1-39 Sau3A 38.3 * [+ or -] 10.7 131 14.9 [+ or -] 10.1 1-39 41.4 * [+ or -] 9.7 77 16.05 [+ or -] 10.0 1-41 * p < 0.05. Table 2. Frequencies of alleles and for each SNP [%(n)]. SNP ALAD1 ALAD2 1-1 1-2 2-2 Msp 95.9 4.1 92.6 (226) 6.6 (16) 0.8 (2) Rsa 46.7 53.3 23.1 (56) 47.1 (114) 29.8 (72) Rsa39488 46.7 53.3 23.2 (56) 46.9 (113) 29.9 (72) HpyCH4 94.2 5.8 88.4 (191) 11.6 (25) 0 HpylV 85.2 14.8 73 (173) 24.5 (58) 2.5 (6) Sau3A 76.9 23.1 62.6 (137) 28.8 (63) 8.7 (19) The distribution of genotypes and alleles within each SNP has been proven to follow the Hardy-Weinberg equilibrium. Table 3. Mean of exposure indices and renal parameters. Biologic parameter n (a) Mean [+ or -] SD Minimum-maximum PbB ([micro]g/dL) (b) 247 24.4 [+ or -] 13.6 2.0-66.9 ALAU (mg/g Cr) 236 0.95 [+ or -] 1.55 0.34-3.63 S[alpha]1m (mg/L) 241 41.8 [+ or -] 1.29 20.84-74.25 S[beta]2m (mg/L) 244 1.45 [+ or -] 1.31 0.77-2.85 NAG (U/g Cr) (c) 236 2.82 [+ or -] 0.18 0.10-7.60 U[alpha]1m (mg/g Cr) 244 6.82 [+ or -] 1.91 1.29-27.32 Ualb (mg/g Cr) 239 7.06 [+ or -] 1.73 1.23-34.12 U[beta]2m (mg/g Cr) 237 0.19 [+ or -] 2.30 0.02-1.81 URBP (mg/g Cr) 239 0.16 [+ or -] 1.95 0.03-0.86 Biologic parameter Range PbB ([micro]g/dL) (b) 64.9 ALAU (mg/g Cr) 3.30 S[alpha]1m (mg/L) 53.42 S[beta]2m (mg/L) 2.09 NAG (U/g Cr) (c) 7.50 U[alpha]1m (mg/g Cr) 26.03 Ualb (mg/g Cr) 32.89 U[beta]2m (mg/g Cr) 1.79 URBP (mg/g Cr) 0.83 Cr, creatinine. Data are geometric mean, except as indicated. (a) Numbers differ between tests because some samples were too small for all the tests to be performed. (b)Arithmetic mean. (c) Square root mean. Table 4. Mean exposure indices and renal parameters by SNP genotypes. HpyCH4 HpyIV Msp Adjusted mean (a) 1-1 1-2 1-1 1-2/2-2 1-1 PbB ([micro]g/dL) (b) 22.68 17.89 23.32 20.52 22.68 ALAU (mg/g Cr) 1 1 0.954 ** 1.150 ** 1.01 S[alpha]1m (mg/L) 42.02 43.9 42.32 42.02 42.21 S[beta]2m (mg/L) 1.41 1.43 1.40 1.4 1.41 NAG (U/g Cr) (c) 2.81 2.46 2.78 2.77 2.77 URBP (mg/g Cr) 0.15 0.13 0.15 0.15 0.15 U[beta]2m (mg/g Cr) 0.18 0.21 0.18 0.19 0.18 Ualb (mg/g Cr) 7.35 6.92 7.374 * 7.750 * 7.29 U[alpha]1m (mg/g Cr) 6.21 5.29 6.15 6.07 6.11 Msp Rsa Rsa39488 Adjusted mean (a) 1-2/2-2 1-1 1-2 2-2 1-1 PbB ([micro]g/dL) (b) 17.89 21.13 21.89 24.22 21.87 * ALAU (mg/g Cr) 0.95 1.07 0.98 0.99 1.03 S[alpha]1m (mg/L) 40.97 42.23 41.99 42.24 42.29 S[beta]2m (mg/L) 1.49 1.36 1.43 1.4 1.37 NAG (U/g Cr) (c) 2.76 3.12 2.73 2.66 3.07 URBP (mg/g Cr) 0.14 0.16 0.14 0.14 0.16 U[beta]2m (mg/g Cr) 0.24 0.18 0.19 0.18 0.18 Ualb (mg/g Cr) 7.19 7.37 6.84 8.10 7.55 U[alpha]1m (mg/g Cr) 6.8 6.49 5.69 6.58 6.5 Rsa39488 Sau3 Adjusted mean (a) 1-2 2-2 1-1 1-2/2-2 PbB ([micro]g/dL) (b) 20.43 * 25.54 * 23.22 20.8 ALAU (mg/g Cr) 1.02 0.96 0.93 ** 1.08 ** S[alpha]1m (mg/L) 42.04 42.58 42.04 42.6 S[beta]2m (mg/L) 1.42 1.46 1.4 1.39 NAG (U/g Cr) (c) 2.81 2.51 2.93 * 2.53 * URBP (mg/g Cr) 0.14 0.14 0.15 0.14 U[beta]2m (mg/g Cr) 0.17 0.2 0.18 0.18 Ualb (mg/g Cr) 6.98 7.79 7.6 6.79 U[alpha]1m (mg/g Cr) 5.72 6.53 5.89 6.36 Data are geometric means adjusted for age, exposure duration, gender, BMI, and PbB, unless otherwise specified. (a)Adjusted for age, exposure duration, gender, and BMI. (b) Arithmetic mean. (c) Square root mean. * p < 0.05. ** p < 0.01. Table 5. Models of HpyCH4 regression by renal parameters. Variable [beta] (95% Cl) p-Values Log URBP [R.sup.2] = 0.1473 PbB 0.016 (0.006, 0.027) 0.002 HpyCH4 -0.02 (-0.140, 0.100) 0.742 HpyCH4 x PbB -0.012 (-0.022, -0.003) 0.008 Log U[alpha]1m [R.sup.2] = 0.2366 PbB 0.018 (0.008, 0.027) < 0.001 HpyCH4 -0.067 (-0.177, 0.043) 0.233 HpyCH4 x PbB -0.014 (-0.022, -0.005) 0.002 Log U[beta]2m [R.sup.2] = 0.1417 PbB 0.029 (0.016, 0.042) < 0.001 HpyCH4 0.047 (-0.103, 0.197) 0.537 HpyCH4 x PbB -0.024 (-0.035, -0.012) < 0.001 Log Ualb [R.sup.2] = 0.1417 PbB 0.014 (0.005, 0.023) 0.002 HpyCH4 0.000 (-0.103, 0.103) 0.998 HpyCH4 x PbB -0.011 (-0.019, -0.003) 0.007 CI, confidence interval. The model adjusts for age, gender, BMI, and exposure duration. 0 represents the HpyCH4 1-1; 1 represents HpyCH4 1-2. The reference group is HpyCH4 1-1; the [beta]-coefficient of PbB is the slope for the association between PbB and renal parameters in participants with this genotype. The corresponding slope in workers with ALAD 1-2 is the sum of the [beta]-coefficient of PbB and that of HpyCH4 x PbB in each model (i.e., in the URBP model, the [beta]-coefficient of PbB for HpyCH4 1-1 group is 0.016; the corresponding [beta] for HpyCH4 1-2 is 0.004 = 0.016 + -0.012). p-Values for the HpyCH4xPbB reflect the statistical significance of the difference between the slopes of the regression line for HpyCH4 1-1 and for HpyCH4 1-2 workers.