In this study, overall lead exposure was associated with an increased risk of RCC, and this risk was modified by some ALAD genotypes. The increased risk associated with lead exposure was highest among subjects that had heterozygous or homozygous variants of the rs8177796 polymorphism and those with the common genotype at SNPs rs8177796 and rs2761016. We found a suggestion of decreased risk among participants with the rs2761016 homozygous variant who were exposed to lead. In addition to finding that occupational lead exposure increased RCC risk, we observed some ALAD variants alone altered cancer risk, independent of lead exposure. Confirmation of our findings will require replication in other large sufficiently powered studies with extensive lead exposure data.
If the risk of RCC is truly increased in individuals with ALAD
polymorphisms, the question of mechanism depends on whether the association is dependent upon exogenous chemical exposures that disrupt the heme synthesis pathway or whether it occurs independently of exposure. The ALAD polymorphisms may encode an enzyme that is less active that the wild type, resulting in an accumulation of 5-aminolevulinic acid (ALA), a precursor thought to be genotoxic [
, reviewed in 
]. However, ALAD enzymatic activity has been shown to not be significantly different 
when comparing SNPs that result in changes in the translated sequence such as rs1800435 (K68N). It is possible that the increased risk of RCC in ALAD
genotypic variants observed is due in part to exogenous chemicals that alter the heme synthesis pathway. Inhibition of ALAD enzymatic activity has been reported for multiple chemicals, including trichloroethylene, bromobenzene, styrene, and lead 
. Polymorphic differences for enzyme inhibition have been most notably studied for lead. Individuals with the polymorphism at position 177 leading to a G→C transversion (rs1800435) results in three isozymes with different affinities for lead binding 
. The homozygous variant has a higher binding affinity for lead and has been associated with increased blood lead levels 
. The biological relevance of this alteration in lead binding is currently being debated in the literature. Studies have suggested that carriers of the G177C polymorphism are more susceptible to lead toxicity 
. Other studies suggest that the enhanced ability for the ALAD isozyme to bind lead actually confers a protective effect by sequestering circulating lead, slowing its accumulation in the bone marrow 
. In this study, we observed a decrease in risk among those exposed to lead who carry the C allele at rs1800435(K68N)
compared to the wildtype G allele, however the number of cases did not provide a stable estimate.
The observation that individuals with the rs8177796 homozygous minor allele have an increased risk of RCC independently of lead exposure may be due to alterations in transcription. The SNP lies within an intronic region; however it is plausible that the polymorphism alters transcription of the wild-type ALAD, resulting in translation of a less active ALAD isozyme. It is also possible that the risk associated with the rs8177796 wild type variant does not represent a change in ALAD, but instead is in high linkage equilibrium with a biologically significant polymorphism that was captured by our tag SNP, but was not part of our examination. In contrast, we observed a decrease in RCC risk and significant interaction with lead exposure for the rs2761016 polymorphism. Similar to rs8177796, this intronic SNP may be altering ALAD activity, or could simply be tagging a region harboring an unidentified polymorphism. Additional genotyping to identify functional variants and in vitro analysis are needed to further explore the impact these intronic polymorphisms on ALAD activity.
The question of renal cancer risk associated with ALAD
genotype has not been previously addressed in the literature. Recent work on ALAD polymorphsims and risk of brain tumors suggests an increased risk for meningioma among participants with the ALAD
G177C homozygous genotype 
. Schober et al. reported an increased risk of all cause, cardiovascular, and cancer mortality 
associated with blood lead levels as low as 5–9 ug/dL 
; however this study did not analyze the role of the ALAD genotype. We did not observe a significant change in RCC risk among participants with the G177C major allele overall or among lead exposed subjects. This variant was quite rare in this population and the number of overall cases exposed to lead was small, there-by under powering this gene-exposure interaction analysis. We chose to evaluate polymorphisms in ALAD
based on the a priori
biological and functional considerations, not by screening a large number of associations. Nonetheless, the possibility that our findings are due to chance cannot be ruled out. The results should be considered as hypothesis generating and require confirmation by replication in other studies.
A limitation of a hospital-based case-control study of occupational exposures is that the distribution of exposed participants might not be representative of the underlying healthy population. We attempted to address this issue by recruiting controls with a wide range of disease diagnoses. Apart from the neurological conditions, diseases reported among controls in our study are not known to be associated with lead exposure. It is thought that hospitalized patients may have different smoking patterns compared to the general population. A recent meta analysis reported the association between smoking and RCC risk is weakest among hospital-based studies 
. We attempted to control for possible selection bias by excluding controls with smoking-related diseases, however the high number of smokers among our control population may indicate a bias in our study. Given multiple centers and countries were used in our study, the potential for population stratification exists; however we found no evidence of heterogeneity. It is possible that population stratification remains, but this is unlikely in European populations 
. The lack of environmental measurements of lead exposure is an additional limitation of our study, which relied upon retrospective recall by study participants regarding their work history and other risk factors. However, since both cases and controls were hospitalized patients, any bias in recall would likely be non-differential with respect to exposure, attenuating the observed risk. Residual confounding by environmental lead exposure is also a limitation. Exposure misclassification is a concern in studies based on retrospective assessment, potentially causing us to underestimate risk if it is non-differential between cases and controls. Finally, the small number of cases exposed to lead and carrying any one ALAD
variant restricts the statistical power of our analysis.
The strengths of this study are the high participation rates thus minimizing the potential for selection bias. The large sample size provided sufficient statistical power to evaluate small associations between genotype (with a prevalence of at least 10%) and risk. However, due to the low exposure prevalence (~6%), our power to detect gene-exposure interactions was limited. Our use of job-specific questionnaire models to collect individual, detailed exposure information and local, expert-based exposure assessments to evaluate exposure histories is considered a superior approach for retrospective assessment of occupational exposures in community-based studies 
. Although we had limited power for evaluating risk with respect to lead exposure and ALAD
homozygous minor alleles (particularly those SNPs with suggested functional relevance), this study is one of the largest case-control studies of RCC and occupational lead exposure to date. In addition to the small percentage of RCC can be explained by familial syndromes including von Hippel-Lindau and hereditary papillary renal carcinoma roughly 50% of RCC incidence is thought to be associated with obesity, hypertension, and smoking 
. The cause of the remaining half of incident cases remains unknown. Therefore, this study was designed to assess occupational and genetic factors in relation to RCC risk in a region with the highest incident rates worldwide 
. To clarify the role of lead in the observed relationship between ALAD
variants and risk of RCC, it will be important to conduct detailed, lead exposure assessments to evaluate how lead exposure; in combination with ALAD
genetic variants could alter cancer risk in other study populations.