We conducted a nested case-control study within the ATBC Study to examine the association between one-carbon metabolism biomarkers and risk of renal cell carcinoma. To our knowledge, this study is the first to examine biomarkers of one-carbon metabolism in association with RCC. We observed a significant increased risk of RCC for men with low folate status, defined as serum folate concentrations near or below 6.6 nmol/L and corresponding to the lowest quartile of serum folate in our population. A possible inverse association was observed for serum folate when analyzed by quartiles, but there was no evidence of a linear trend. There was no evidence of an association with RCC for vitamin B6, vitamin B12, cysteine, riboflavin or homocysteine.
The apparent threshold effect for folate occurred near 6.6 nmol/L, which corresponds to the concentration associated with clinical deficiency and risk of megaloblastic anemia [33
]. A sensitivity analysis indicated that the effect near 6.6 nmol/L is a threshold and not simply an artifact of our quartile-based analysis. A threshold effect is biologically plausible given the hypothesized mechanism of action, which is inadequate methylation and/or DNA synthesis due to reduced activity of the one-carbon metabolism pathway. A certain minimum level of folate may be necessary to allow the one-carbon transfer required for maintenance of adequate DNA synthesis and/or methylation. Inadequate folate availability could disrupt these processes and increase cancer risk. Increasing the available folate above the minimum required level may not result in a further decrease in risk because an adequate level of one-carbon transfer has already been attained. If this is the case, the lack of a linear trend across quartiles of serum folate is not surprising. It is increasingly being recognized that the effects of folate exposure on carcinogenesis are complicated and likely vary by both dosage and timing. The idea of a nonlinear threshold relationship with increasing folate beyond an optimum exposure has been previously discussed, particularly in the context of colorectal cancer [34
]. Although we observed an increased risk of RCC among subjects with the lowest concentrations of serum folate, we did not observe any association between serum homocysteine and RCC risk. The lack of association is noteworthy because elevated serum homocysteine can be an indicator of folate deficiency, although other metabolic pathways also influence homocysteine concentrations and the correlation between serum homocysteine and serum folate was modest (-0.28) in our study.
Some cohort studies, but not all, have found associations between higher fruit and vegetable intake and reduced risk of RCC [7
]. A recent pooled analysis of 13 prospective studies, including the ATBC study, found a significant protective effect of increased fruit and vegetable consumption [12
]. Fruits and vegetables are primary sources of dietary folate, particularly in studies conducted prior to folic acid fortification or in non-fortified populations such as Finland [36
], so the observed associations may support a role of folate intake. However, fruits and vegetables are also sources of numerous other components thought to impact health and carcinogenesis, so other non-folate explanations for the associations are possible. A recent study that examined only the ATBC population did not find an association between fruit and vegetable consumption and RCC risk [11
], possibly due to lower power relative to the pooled analysis or lower intake of fruits and vegetables compared to other prospective studies [12
]. A role for one-carbon metabolism in RCC risk is supported by a candidate gene study that reported significant associations between single nucleotide polymorphisms in one-carbon metabolism genes and RCC risk [24
]. The MTHFR gene is a key regulator of one-carbon metabolism, and a variant allele known to decrease MTHFR activity [39
] was associated with increased risk of RCC in a population from Central and Eastern Europe. Additional polymorphisms in the TYMS gene were also associated with RCC risk, particularly among people with low folate intake.
A major strength of our study is that it is prospective, with all data being collected prior to disease diagnosis. The use of serum biomarkers for the one-carbon factors may be a better indication of absorbed and biologically active dose compared to self-reported nutrient intake. Serum folate tends to reflect short-term folate balance [40
], but still has good predictive value as an indicator of systemic folate status [33
]. The prospective nature of the exposure assessment minimizes the potential of biomarker concentrations being altered by existing cancers (reverse causation). Recall bias is also minimized because information on risk factors and diet were collected at baseline, and biased control selection is not an issue since cases and controls were selected from the same study cohort. Our study also included a long follow-up time, allowing identification of a relatively large number of RCC cases and examination of the effect of biomarker concentrations over an extended period. Finland does not have mandatory fortification of grain products with folic acid, so our study population included subjects with low folate status that would rarely be seen in fortified populations. The mean (± standard error) serum folate concentration for subjects in our study was 8.6 (± 0.2) nmol/L, compared to 26.9 (± 0.5) nmol/L in a United States population after fortification [38
]. Finally, we were able to investigate the impact of multiple biomarkers of one-carbon metabolism rather than just folate alone.
Our study was conducted in a cohort of male Finnish smokers, and therefore may not be generalizable to other populations. Smoking is associated with a modest increased risk of RCC [3
], and smokers have been found to have lower serum and red blood cell folate concentrations than non-smokers [42
]. The reduced folate status of smokers may be due to lower intake of folate as well as greater body requirements due to the effects of tobacco smoke [42
]. Folate intake in the ATBC cohort was relatively low, with 75% of control subjects having an intake below the U.S. Dietary Reference Intake of 400 ug/day. Although we adjusted for smoking in all models (both number of cigarettes smoked per day and years of smoking), the possibility of residual confounding remains. However, there was no significant interaction for the folate association by smoking dose or duration, and an analysis restricted to subjects who reported smoking 20 or more cigarettes per day showed similar associations between folate and RCC risk (data not shown). In addition, adjustment for smoking cessation during the ATBC trial did not change the risk estimates for serum folate and RCC. The role of folate and one-carbon metabolism in RCC risk needs to be further examined in populations including nonsmokers and women. However, studies conducted in populations with folic acid fortification of grain products may not be able to examine effects of deficient folate status. Further limitations of our study include the fact that assessment of the biomarkers in this study occurred at a single point in time, so they may not have captured lifetime exposure and may have changed over the course of the long follow-up period. Finally, serum biomarkers may not correlate with tissue-specific concentrations in the kidney.
In conclusion, we found that low serum folate concentrations were associated with a non-significant increased renal cell carcinoma risk. Analysis of a threshold effect near the level of folate deficiency showed a moderate and statistically significant increased risk of RCC. There was no evidence of a dose-response trend across quartiles, so the association with serum folate appears to be restricted to concentrations considered deficient. Given the mandatory fortification of grain products with folic acid in the United States and the resulting increase in population folate status (< 1% of the U.S. population has deficient concentrations of serum folate) [45
], folate deficiency is unlikely to play a large role in RCC risk in the United States and other fortified populations. However, folate may be a risk factor in other parts of the world where folic acid fortification is not widespread and deficient status is more prevalent. We found no associations for the other one-carbon biomarkers examined (vitamin B6
, vitamin B12
, cysteine, riboflavin and homocysteine). A role for one-carbon metabolism in renal cancer development is biologically plausible, but epidemiologic evidence regarding such a role is limited. Further studies are needed to more clearly elucidate the relationship between one-carbon metabolism and renal cell carcinoma, particularly for folate intake and status in other populations.